‎⁨حيوانية 4⁩ 2.pdf

First lecture First semester/2021---2022 Class:4th students Animal Tissue Culture 1- The Historical Background Tissue culture enters its second century, and was devised at the beginning of the twentieth century. [Harrison, 1907; Carrel, 1912] wh...

First lecture First semester/2021---2022 Class:4th students Animal Tissue Culture 1- The Historical Background Tissue culture enters its second century, and was devised at the beginning of the twentieth century. [Harrison, 1907; Carrel, 1912] who studying:  the behavior of animal cells, with undisaggregated fragments of tissue, and  growth was restricted to the radial migration of cells from the tissue fragment with occasional mitoses in the outgrowth. [Rous & Jones, 1916], was first demonstrated the Disaggregation of explanted cells and subsequent plating out of the dispersed cells by, although passage was more often by surgical subdivision of the culture by Fischer, Carrel, and others, to generate what were then termed cell strains. [Fischer, 1925], culture of cells from and within such primary explants of tissue dominated the field for more than 50 years, it is not surprising that the name ‘‘tissue culture’’ has remained in use as a generic term, despite the fact that most of the explosive expansion in the field in the second half of the twentieth century was made possible by the use of dispersed cell cultures. 1  [Sanford et al., 1948] was cultured L929 was the first cloned cell strain, isolated by capillary cloning from mouse L-cells. [Dulbecco, 1952], after the 1950s that trypsin became more generally used for subculture, following procedures described by Dulbecco to obtain passaged monolayer cultures for viral plaque assays and the generation of a single cell suspension by trypsinization, which facilitated the further development of single cell cloning. Gey et al., 1952 established the first continuous human cell line, HeLa; this was subsequently cloned by Puck [Puck & Marcus, 1955] when the concept of an X- irradiated feeder layer was introduced into cloning. [Parker, 1961] using antibiotic to prevent contaminations, though tissue culture became more widely used at this time because of the introduction of antibiotics, which facilitated long-term cell line propagation, then resistant contaminations. [Ham, 1963, 1965] after controlling on resistant contaminations of culture, led ultimately to the development of serum-free media In 1970 Kohler and Milstein produced an antibody –secreting hybridoma. Sato in 1980 developed serum free media from hormones and growth factors, was produced "human insulin from bacteria" 1990 Recombinant tissue plasminogen activator (t-PA) was produced from animal cells. Terms of Tissue culture o Tissue culture is used as a generic term to include:  organ culture and  cell culture. The term "organ culture" will always imply a three-dimensional culture of undisaggregated tissue retaining some or all of the histological features of the tissue in vivo. "Cell culture" refers to a culture derived from dispersed cells taken from original tissue, from a primary culture, or from a cell line or cell strain by enzymatic, mechanical, or chemical disaggregation. o The term histotypic culture implies that cells have been reaggregated or grown to recreate a three-dimensional structure with tissue like cell density, for example, by cultivation at high density in a filter well, by perfusion and overgrowth of a monolayer in a flask or dish, by reaggregation in suspension (Where? )..over agar or in real or simulated zero gravity, or by infiltration of a three-dimensional matrix such as collagen gel. 2 o Organotypic implies the same procedures but recombining cells of different lineages, such as epidermal keratinocytes in combined culture with dermal fibroblasts, in an attempt to generate a tissue equivalent. Early experiments in tissue culture: Why Harrison chose the frog for his experiment? Harrison chose the frog as his source of tissue, because: 1- it was a cold-blooded animal, and consequently incubation was not required. 2- Tissue regeneration is more common in lower vertebrates, he perhaps felt that growth was more likely to occur than with mammalian tissue. The stimulus from medical science carried future interest into warm-blooded animals, in which both normal development and pathological aberrations are closer to that found in humans. The accessibility of different tissues, many of which grew well in culture, made the embryonated hen’s egg a favorite choice (second chose of organism), but the development of experimental animal husbandry, particularly with genetically pure strains of rodents, brought mammals to the forefront as the favorite material. Although chick embryo tissue could provide a diversity of cell types in primary culture, rodent tissue had the advantage of producing continuous cell lines and a considerable repertoire of transplantable tumors. The demonstration that human tumors could also give rise to continuous cell lines, such as HeLa [Gey et al., 1952], encouraged interest in human tissue, that helped later by the classic studies on the finite life span of cells in culture and the requirement of virologists and molecular geneticists to work with human material. The development of transgenic mouse technology together with the well- established genetic background of the mouse, has added further impetus (stimulus) to the selection of this animal as a favorite species. The cultivation of human cells received a further stimulus when a number of different serum free selective media were developed for specific cell types, such as:  epidermal keratinocytes,  bronchial epithelium, and  vascular endothelium. These formulations are now available commercially, although the cost remains high relative to the cost of regular media. 3  Procedures for handling nonmammalian cells have tended to follow those developed for mammalian cell culture, although a limited number of specialized media are now commercially available for fish and insect cells. 2- TYPES OF TISSUE CULTURE There are three main methods of initiating a culture: (1) Organ culture implies that the architecture characteristic of the tissue in vivo is retained, at least in part in the culture. Toward this end the tissue is cultured at the liquid–gas interface (on a raft, grid, or gel), which favors the retention of a spherical or three- dimensional shape. (Next figure -2). Because of the retention of histological interactions found in the tissue from which the culture was derived, organ cultures tend to retain the differentiated properties of that tissue. They do not grow rapidly (cell proliferation is limited to the periphery of the explants and is restricted mainly to embryonic tissue) and hence cannot be propagated; each experiment requires fresh explanations. (2) In primary explant culture a fragment of tissue is placed at a glass (or plastic)–liquid interface, where, after attachment, migration is promoted in the plane of the solid substrate. (3) Cell culture implies that the tissue, or outgrowth from the primary explant, is dispersed (mechanically or enzymatically) into a cell suspension, which may then be cultured as an adherent monolayer on a solid substrate or as a suspension in the culture medium. 4  The formation of a cell line from a primary culture implies (involve) an increase in: (1) The total number of cells over several generations (population doublings) (2) The ultimate predominance of cells or cell lineages with a high proliferative capacity, resulting lately in (3) Increasing a degree of uniformity in the cell population. The line may be characterized, and the characteristics will apply for most of its finite life span. The sources or beginning of culture derivation of: Continuous (or ‘‘established,’’ as they were once known) cell lines usually implies a genotypic change, or transformation, and the cell formation is usually accompanied by  an increased rate of cell proliferation and  a higher plating efficiency. a Cell strain is known to cells are selected from a culture, by  cloning or by  some other method, the subline is known cell strain. Cell lines or cell strains a detailed characterization is then implied may be propagated as an adherent monolayer or in suspension. Types of cell culturing:  Monolayer culture "an adherent monolayer" Monolayer culture signifies that the cells are grown attached to the substrate. Anchorage dependence means that attachment to the substrate is a prerequisite for cell proliferation, It is the mode of culture common to most normal cells, with the exception of hematopoietic cells.  Suspension cultures are derived from cells that can survive and proliferate without attachment (anchorage independent); this ability is restricted to hematopoietic cells, transformed cell lines, and transformed cells from malignant tumors. It can be shown, however, that a small proportion of cells that are capable of proliferation in suspension exist in many normal tissues. Many workers have attempted to reconstitute three dimensional cellular structures. Because cultured cell lines lack the retention of cell–cell interaction and cell–matrix interaction afforded by organ cultures For this reason such developments have required the introduction, or at least redefinition, of certain terms. Represented in:  Histotypic culture, or histoculture (use histotypic culture), has come to mean the high-density, or ‘‘tissue-like,’’ culture of one cell type, Whereas 5   organotypic culture implies the presence of more than one cell type interacting, as the cells might, in the organ of origin. Organotypic culture, defined populations of homogeneous and potentially genetically and phenotypically defined cells and an opportunity to create differentiated populations of cells suitable for grafting. 3- ADVANTAGES OF TISSUE CULTURE 1- Control of the Environment The two major advantages of tissue culture are the ability to control: A- The physiochemical environment (pH, temperature, osmotic pressure, and O2 and CO2 tension), which has to be controlled very precisely. B- The physiological conditions, which have to be kept relatively constant. However, the physiological environment cannot always be defined where cell lines still require supplementation of the medium with serum or other poorly defined constituents. These supplements such as hormones and other stimulants and inhibitors. The identification of some of the essential components of serum, together with a better understanding of factors regulating cell proliferation, has made the replacement of serum with defined constituents feasible. The role of the extracellular matrix (ECM) is important but similar to the use of serum—that is, the matrix is often necessary, but not always precisely defined. 2 - Characterization and Homogeneity of Samples Tissue samples are invariably heterogeneous. Replicates, even from one tissue, vary in their constituent cell types. After one or two passages, cultured cell lines assume a homogeneous (or at least uniform) constitution, as the cells are randomly mixed at each transfer and the selective pressure of the culture conditions tends to produce a homogeneous culture of the most vigorous cell type. Hence, at each subculture, replicate samples are identical to each other, and the characteristics of the line may be perpetuated (preserved) over several generations, or even indefinitely if the cell line is stored in liquid nitrogen. Furthermore, the availability of stringent tests for cell line identity and contamination means that preserved stocks may be validated for future research and commercial use. 4 --- LIMITATIONS (Disadvantage) 1. Expertise Culture techniques must be carried out under strict aseptic conditions because animal cells grow much less rapidly than many of the common contaminants, such as bacteria, molds, and yeasts. Furthermore, unlike microorganisms, cells from multicellular animals : 6  a- do not normally exist in isolation and consequently are not able to sustain an independent existence without the provision of a complex environment simulating blood plasma or interstitial fluid. b- These conditions imply a level of skill and understanding on the part of the operator in order to appreciate the requirements of the system and – c-to diagnose problems care must be taken to avoid the recurrent problem of cross-contamination. 2. Quantitation is therefore more difficult, and the amount of material that may be cultured is limited by the dimensions of the explant (≥1 mm3) and the effort required for dissection and setting up the culture. Cell cultures may be derived from primary explants or dispersed cell suspensions. A monolayer or cell suspension with a significant growth fraction may be dispersed by enzymatic treatment or simple dilution and reseeded, or subcultures, into fresh vessels. This constitutes a subculture or passage, and the daughter cultures so formed are the beginnings of a cell line. 3. Dedifferentiation and Selection Loss of the phenotypic characteristics typical of the tissue from which the cells had been isolated were observed by workers in cell line propagation. This effect was blamed on dedifferentiation, (a reversal of differentiation but later shown to be largely due to the overgrowth of undifferentiated cells of the same or a different lineage). The development of serum-free selective media has now made the isolation of specific lineages possible, and it can be seen that under the right conditions, many of the differentiated properties of these cells may be restored. 4. Origin of Cells If differentiated properties are lost, for whatever reason, it is difficult to relate the cultured cells to functional cells in the tissue from which they were derived. Stable markers are required for characterization of the cells; in addition, the culture conditions may need to be modified so that these markers are expressed. Unfortunately, many cell lines have been misidentified due to: cross-contamination or errors in stock control in culture or in the freezer 5. Instability Instability is a major problem with many continuous cell lines, resulting from their unstable aneuploidy chromosomal constitution. Even with short-term cultures of untransformed cells, heterogeneity in growth rate and the capacity to differentiate within the population can produce variability from one passage to the next. 7 5 - MAJOR DIFFERENCES IN VITRO Most of the differences in cell behavior between cultured cells (in vitro) and their counterparts in vivo stem from the dissociation of cells from a three-dimensional geometry and their propagation on a two dimensional substrate. Specific cell interactions characteristic of the histology of the tissue are lost. As the growth fraction of the cell population increases, the cells spread out, become mobile, and, in many cases, start to proliferate. When a cell line forms, it may represent only one or two cell types, and many heterotypic cell–cell interactions are lost. such as: a-Culture environment also lacks the several systemic components involved in homeostatic regulation in vivo, principally those of the nervous and endocrine systems) b-Cellular metabolism may be more constant in vitro than in vivo, but may not be truly representative of the tissue from which the cells were derived. c- The low oxygen tension due to the lack of oxygen transporter (hemoglobin) results in energy metabolism in vitro occurring largely by glycolysis; although the citric acid cycle is still functional, it plays a lesser role. 16 October 2021 Asst. Prof. Maha Al- Tati Prof. Dr.Mahfoodha Abbas Umran ‫ العبارة الموجودة داخل التضليل الرصاصي كهذه هي لتوضيح معلومة وغير مطلوبة‬:‫مالجظة‬ 8  Second lecture First semester/2022--2023 4th Class Animal tissue cultures 1-INITIATION OF A PRIMARY CELL CULTURE To initiation of primary culture there are four stages to consider: (1) Acquisition (Gaining) of the sample, (2) Isolation of the tissue, (3) Dissection and/or disaggregation, (4) Culture after seeding into the culture vessel. After isolation, a primary cell culture may be obtained either by: 1- allowing cells migrate out from fragments of tissue adhering to a suitable substrate or 2-disaggregating the tissue mechanically or enzymatically to produce a suspension of cells, some of which will attach to the substrate. It (attachment) appears to be essential for most normal untransformed cells, with the exception of hematopoietic cells and stem cells, to attach to a flat surface in order to survive and proliferate with maximum efficiency. Transformed cells, however, particularly cells from transplantable animal tumors, are often able to proliferate in suspension. What and how Enzymes used in Disaggregation? The enzymes used: alone most frequently for tissue disaggregation are crude preparations of trypsin, collagenase, elastase, pronase, Dispase, DNase, and hyaluronidase, or in various combinations, such as: a- Elastase and DNase, b - collagenase with Dispase, c- collagenase with hyaluronidase There are other, nonmammalian enzymes, such as: Trypzean (Sigma), a recombinant, maize derived, trypsin, TrypLE (Invitrogen), recombinant microbial, and Accutase malso available for primary disaggregation Collagenase used to digest the extracellular matrix, and DNase is used to disperse DNA released from lysed cells; DNA tends to impair (harm, or damage) proteolysis and promote reaggregation. Care should be taken when combining enzymes, for example DNase should be added after trypsin has been removed, as the trypsin may degrade the DNase. 1 2- Common Features of Disaggregation Although each tissue may require a different set of conditions, certain requirements are shared by most of them: (1) Fat and necrotic tissues are best removed during dissection. (2) The tissue should be chopped finely with sharp scalpels to cause minimum damage. (3) Enzymes used for disaggregation should be removed subsequently by gentle centrifugation. (4) The concentration of cells in the primary culture should be much higher than that normally used for subculture because the proportion of cells from the tissue that survives in primary culture may be quite low. (5) A rich medium, such as Ham’s F12, is preferable to a simple medium, such as Eagle’s MEM, and if serum is required, fetal bovine often gives better survival than does calf or horse (6) Embryonic tissue disaggregates more readily, yields more viable cells, and proliferates more rapidly in primary culture than does adult tissue. 3- ISOLATION OF THE TISSUE Work with human biopsies or fetal material usually requires the consent (agreement) of the local ethical committee and the patient and/or his or her relatives. For example, in the United Kingdom, the use of embryos or fetuses beyond 50% gestation or incubation is regulated under the Animal Experiments Safety Note. Work with human tissue should be carried out at Containment Level 2 in a Class II biological safety cabinet. Work with human needs an attempt should be made: To sterilize the site of the resection with 70% alcohol if the site is likely to be contaminated (e.g., skin). Remove the tissue aseptically and transfer it to the tissue culture laboratory in dissection or collection medium as soon as possible. Do not dissect animals in the tissue culture laboratory, as the animals may carry microbial contamination. If a delay in transferring the tissue is unavoidable, it can be held at 4◦C for up to 72 h, although a better yield will usually result from a quicker transfer. Embryonic or fetal animals that are more than half term may require specified methods of humane killing before dissection. (What are the tissues used for cells isolation)? 3-A: Mouse Embryo 2  Mouse embryos are a convenient source of cells for undifferentiated mesenchymal cell cultures. These cultures are often referred to as ‘‘mouse embryo fibroblasts’’ and used as feeder layers. Full term is about 19 to 21 days, depending on the strain. The optimal age for preparing cultures from a whole disaggregated embryo is around 13 days, when the embryo is relatively large but still contains a high proportion of undifferentiated mesenchyme, isolation and handling embryos beyond 50% full-term may require a license. So 9- or 10-day embryos may be preferable. Why this period is preferable? Answer: the amount of tissue recovered from these embryos will be substantially less, a higher proportion of the cells will grow. To obtain specific cells exist in organs, Dissection of individual organs is easier at 13 to 14 days, and most of the organs are completely formed by the 18th day. 3-B: Chick Embryo Chick embryos are easier to dissect, as they are larger than mouse embryos at the equivalent stage of development. Like mouse embryos, chick embryos are used to provide predominantly mesenchymal cell primary cultures for cell proliferation analysis, to provide feeder layers, and as a substrate for viral propagation. Because of their larger size, it is easier to dissect out individual organs to generate specific cell types, such as hepatocytes, cardiac muscle, and lung epithelium. 3-C: Human Biopsy Material Handling human biopsy material presents certain problems that are not encountered with animal tissue. It usually is necessary to: (1) obtain consent from the hospital ethical committee, (2) from the attending physician or surgeon, and (3) from the donor or patient or the patient’s relatives. Biopsy sampling is usually performed for diagnostic purposes, and hence the needs of the pathologist must be met first. This factor is less of a problem if extensive surgical resection or nonpathological tissue (e.g., placenta or umbilical cord) is involved. The sample of biopsy must be defined in formal collection or storage system must be employed for times by you or someone on your staff in lab. If delivery to your lab is arranged, then there must be a system for receiving specimens. 1-Recording details of the source, 2- Origin of tissue 3-pathology, and 4- Warning (telling) the person who will perform the culture that the specimens have arrived; otherwise, valuable material may be lost or spoiled. 3 4- TYPES OF PRIMARY CULTURE Several techniques have been devised for the disaggregation of tissue isolated for primary culture. These techniques can be divided into: (1) Purely mechanical techniques, involving dissection with or without some form of maceration (softening or soaking). (2) Techniques utilizing enzymatic disaggregation (Fig. 11.5). How the primary culture could be obtained? 4-1: Primary explants are suitable for very small amounts of tissue; enzymatic disaggregation gives a better yield when more tissue is available, and mechanical disaggregation works well with soft tissues and some firmer tissues when the size of the viable yield is not important, or loosely adherent cells are removed from a more fibrous stroma. A- Principle Procedure/ Primary Explanation by Harrison The primary explant technique was the original method developed by Harrison , Carrel , and others for initiating a tissue culture. As originally perform ▪ fragment of tissue was embedded in blood plasma or lymph, ▪ mixed with heterologous serum (induced clotting of the plasma) and embryo extract (supplied nutrients and growth factors), and ▪ placed on a coverslip that was inverted over a concavity slide to stimulated cell migration from the explant. The clotted plasma held the tissue in place, and the explant could be examined with a conventional microscope. This technique is particularly useful for small amounts of tissue, such as skin biopsies, for which there is a risk of losing cells during mechanical or enzymatic disaggregation. Its disadvantages lie in the poor adhesiveness of some tissues and the selection of the more migratory cells in the outgrowth. In practice, however, most cells, particularly embryonic, migrate out successfully. Attaching explants: Both adherence and migration may be stimulated by placing a glass coverslip on top of the explant, with the explant near the edge of the coverslip, or the plastic dish may be scratched through the explants to attach the tissue to the flask. B-How could increase the attachment? Attachment may also be promoted by treating the plastic with polylysine or fibronectin, extracellular matrix, or feeder layers. 4 Asst. Prof. Dr. Maha Al- Tayi Prof. Dr. Mahfoodha Abbas Umran 16/ Oct 2022 5  Third lecture First semester / 2022--2023 4th Class. Animal tissue culture How the primary culture could be obtained? 4-1: Primary explants are suitable for very small amounts of tissue; by disaggregation Enzymatic disaggregation gives a better yield when more tissue is available, and Mechanical disaggregation works well with soft tissues and some firmer (safer or stronger) tissues when the size of the viable yield is not important, or loosely adherent cells are removed from a more fibrous stroma. A- Principle Procedure (Primary Explantation by Harrison) The primary explant technique was the original method developed by Harrison , Carrel , and others for initiating a tissue culture. As originally perform fragment of tissue was embedded in blood plasma or lymph, mixed with: heterologous serum (induced clotting of the plasma) and embryo extract (supplied nutrients and growth factors), and placed on a coverslip that was inverted over a concavity slide to stimulated cell migration from the explant. The clotted plasma held the tissue in place, and the explant could be examined with a conventional microscope. This technique is particularly useful for small amounts of tissue, such as skin biopsies, for which there is a risk of losing cells during mechanical or enzymatic disaggregation. Its disadvantages lie in the poor adhesiveness of some tissues and the selection of the more migratory cells in the outgrowth. In practice, however, most cells, particularly embryonic, migrate out successfully. Attaching explants: Both adherence and migration may be stimulated by placing a glass coverslip on top of the explant, with the explant near the edge of the coverslip, or the plastic dish may be scratched through the explants to attach the tissue to the flask. B-How could increase the attachment? Attachment may also be promoted by treating the plastic with: ▪ Poly lysine or fibronectin, ▪ extracellular matrix, ▪ or feeder layers. 1  Dissociation techniques 4-2: Enzymatic Disaggregation Cell–cell adhesion in tissues is mediated by a variety of homotypic interacting glycopeptides (cell adhesion molecules, or CAMs), some of glycopeptides are: a- Calcium dependent (cadherins) are sensitive to chelating agents such as EDTA. b- Integrin's, which bind to the arginine-glycine-aspartic acid (RGD) motif in extracellular matrix. Intercellular matrix and basement membranes contain ▪ Other glycoproteins, such as fibronectin and laminin, which are protease sensitive, and ▪ Proteoglycans, which are less so but can sometimes be degraded by glycanases such as hyaluronidase or heparinase. There are several rotes of using trypsin: 1- Alone: trypsin 2- Complex solution: of trypsin/EDTA as a starting point, adding or substituting other proteases to improve disaggregation, and deleting trypsin if necessary to increase viability. 3- cocktails of enzymes: Including proteases with cocktails of enzymes, sometimes there is a risk as the resulting proteolysis make inactivate some of the enzymes. 2  Crude trypsin is the most common enzyme used in tissue disaggregation, as it is tolerated quite well by many cells and is effective for many tissues. Residual activity left after washing is neutralized by the serum of the culture medium, or by a trypsin inhibitor (e.g., soya bean trypsin inhibitor) when serum- free medium is used. Mechanical and enzymatic disaggregation of the tissue avoids problems of selection by migration and yields a higher number of cells that are more representative of the whole tissue in a shorter time. However, just as the primary explants technique selects on the basis of cell migration, dissociation techniques will select protease- and mechanical stress- resistant cells. Are all tissues dispersed similarly? Answer: NO Embryonic tissue disperses more readily and gives a higher yield of proliferating cells than newborn or adult tissue. The increasing difficulty in obtaining viable proliferating cells with increasing age is due to several factors, including: 1- The onset of differentiation, 2- an increase in fibrous connective tissue and 3- Extracellular matrix, and 4- A reduction of the undifferentiated proliferating cell pool. When procedures of greater severity are required to disaggregate the tissue (e.g., longer trypsinization or increased agitation), the more fragile components of the tissue may be destroyed. For example: In fibrous tumors, it is very difficult to obtain complete dissociation with trypsin while still retaining viable carcinoma cells. How trypsin and other enzymes is used? Trypsin is used in two conditions: 1-Warm Trypsin The tissue is chopped and stirred in trypsin for a few hours ,It is important to minimize the exposure of cells to active trypsin in order to preserve maximum viability. Hence, when whole tissue is being trypsinized at 37◦C, dissociated cells should be collected every half hour, and the trypsin should be removed by centrifugation and pooled(shared) in medium containing serum. The warm trypsin technique is useful for the disaggregation of large amounts of tissue in a relatively short time, particularly for chopped whole mouse embryos or chick embryos. 3  But warm trypsin technique does not work as well with adult tissue, in which there is a lot of fibrous connective tissue, and mechanical agitation can be damaging to some of the more sensitive cell types, such as epithelium. What enzyme devised in this sample (adult tissue)?..... 2-Cold trypsin The cold trypsin method is particularly suitable for small amounts of tissue, such as embryonic organs. It gives good reproducible cultures from 10- to 13- day chick embryos with evidence of several different cell types characteristic of the tissue of origin. This protocol forms a good exercise for teaching purposes. 3-Other Enzymatic Procedures Disaggregation in trypsin can be damaging (e.g., to some epithelial cells) or ineffective for very fibrous tissue (e.g., such as fibrous connective tissue), So attempts have been made to utilize other enzymes. Because the extracellular matrix often contains collagen, particularly in connective tissue and muscle, 1- collagenase has been the obvious choice for culturing (colon carcinoma); (breast carcinoma), (pancreatic islet cells). 2- Other bacterial proteases, such as Pronase and Dispase have also been used with varying degrees of success. 3- Liberase (Roche) is a cocktail of enzymes (neutral protease with collagenase or thermolysin) that has been used for isolation of: ▪ hepatocytes from liver and ▪ islet cells from pancreas 4- The participation of carbohydrate in intracellular adhesion has led to the use of hyaluronidase and neuraminidase in conjunction with collagenase. [Berry & Friend, 1969] 4- Collagenase This technique is very simple and effective for many tissues: a- embryonic, b-adult, normal, and c- malignant. When collagenase is considering as benefit? It is of greatest benefit when the tissue is either too fibrous or too sensitive to allow the successful use of trypsin. 4  The first disaggregation in collagenase has proved particularly suitable for the culture of human tumors, mouse kidney, human adult and fetal brain, liver, lung, and many other tissues, particularly epithelium The properties of using disaggregation in collagenase are : The process is gentle and requires no mechanical agitation or special equipment. With more than 1 g of tissue, however, it becomes tedious (deadly) at the dissection stage and can be expensive, because of the amount of collagenase required. It will also release most of the connective tissue cells, (heightening or stressing) accentuating the problem of fibroblastic outgrowth, so it may need to be followed by selective culture or cell separation. 4-3: Mechanical Disaggregation The advantage of mechanical disaggregation is the outgrowth of cells from primary explants is a relatively slow process can be highly selective. Enzymatic digestion is rather more labor intensive, although, potentially, it gives a culture that is more representative of the cells in the tissue. As there is a risk of proteolytic damage to cells during enzymatic digestion, Many people have chosen to use mechanical disaggregation: There are many types of mechanical disaggregation for example, 1- spillage: collecting the cells that spill out when the tissue is carefully sliced and the slices scraped; 2- sieving: pressing the dissected tissue through a series of sieves for which the mesh is gradually reduced in size; 3- syringing: forcing the tissue fragments through a syringe (with or without a wide-gauge needle) or simply pipe ting it repeatedly. ❖ This technique (mechanical disaggregation is well and the tissues that are respond well are only soft tissues, such as: spleen, liver, embryonic and adult brain, and some human and animal soft tumors. ❖ The viability of the resulting suspension is lower than that achieved with enzymatic digestion, although the time taken may be very much less. ❖ When the availability of tissue is not a limitation and the efficiency of the yield is not important, it may be possible to produce, in a shorter amount of time, as many viable cells with mechanical disaggregation as with enzymatic digestion, but at the expense of very much more tissue. 5  5- Separation of Viable and Nonviable Cells When tissue is disaggregated and seeded into primary culture, only a proportion of the cells are capable of surviving and generating a primary culture). There are represented in two portion: a-Some cells may not be capable of attachment but yet viable; b- others are nonviable, necrotic or apoptotic. If it is important to do so, the proportion of necrotic and apoptotic cells may be determined viability staining and flow cytometry. Normally nonviable cells are removed at the by: 1- First change of medium. 2- Nonviable cells are gradually diluted out when cell proliferation starts with primary cultures maintained in suspension, 3- Nonviable cells may be removed from the primary disaggregate by centrifuging the cells on a mixture of Ficoll and sodium metrizoate (e.g., Hypaque or Triosil). This technique is similar to the preparation of lymphocytes from peripheral blood). The viable cells collect at the interface between the medium and the Ficoll/metrizoate, and the dead cells form a pellet at the bottom of the tube. Reference: Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Sixth Edition, by R. Ian Freshney Copyright 2010 John Wiley & Sons, Inc. Prof. Dr. Mahfoodha Abbas Umran 23 /Oct. 2022 6  1 Fourth Lecture First semester/2022---2023 4th Class/ Biotechnology Cell types in tissues and culturing 1- After primary culture is growing The cells in a primary culture stop growing a new culture may be established by inoculating some of the cells into fresh medium. This is called sub culturing or passaging. A secondary culture is established after the first passage of the primary culture. The term ‘cell line’ is applied to the cell population that can continue growing through many subcultures be noted that the greatest chance of genetic alteration occurs in the first few passages following the primary culture as cells adapt to a new chemical environment. The chick embryo fibroblasts may grow for around 30 passages before becoming senescent. The passage number of a culture is often recorded as the number of subcultures from the primary source. Q// How long will primary cultures survive? and What is the senescent phase? Hayflick and Moorhead in 1961 studied the growth potential of human embryonic cells. They showed that: Human embryonic cells could be grown continuously through repeated subculture for about 50 generations. After this time, the cells enter a senescent phase and are incapable of further growth. The pattern of growth during the lifespan of these cells illustrates in figure Figure 1.3 Normal and transformed growth of human embryonic cells over an extended number of passages  2 All cells derived from normal animal tissue is a characteristic of a finite growth capacity. The finite number of generations of growth is referred to as the ‘Hayflick limit’ and is a characteristic of the: Cell type, Age and Species of origin ‘Hayflick limit’: The capacity for growth is related to the origin of the cells—those derived from" embryonic tissue have a greater growth capacity than those derived from adult tissue". Each cell appears to have an inherent ‘biological clock 'defined by the number of divisions from the original stem cell. Even if cells are stored by cryopreservation the total capacity for cell division is not altered 2- Cell types in tissues Animal cells are usually defined by the tissue from which they have derived and have characteristic shapes that can be observed and recognized easily through a light microscope. The morphology of the cells commonly grown in culture derived from five main types of animal tissue. These are: 1- Epithelial tissue consists of a layer of cells which cover organs and line cavities; examples include skin and the lining of the alimentary canal. The epithelial cells grow well in culture as a single cell monolayer. 2-Connective tissue forms a major structural component of animals, consisting of a fibrous matrix and including bone cartilage. The tissue contains fibroblasts which are amongst the most widely used cells in laboratory cultures. The cells are spherical when first dissociated by trypsin from the tissue but elongate to a characteristic spindle-shape on attachment to a solid surface. Fibroblasts are bound to the fibrous protein collagen in the connective tissue. Fibroblasts have excellent growth characteristics and have been the ‘favorite’ cells for establishing cultures. Fibroblast and epithelial cells adapt relatively easily to culture and have growth rates with a doubling time of 18–24 hours. 3- Muscle tissue consists of a series of tubules formed from precursor cells to form a multinucleate complex and which also contain the structural proteins (actin and myosin). The precursor cells are myoblasts which are capable of differentiation to form myotubes—a process that can be observed in culture. 4- Nervous tissue consists of characteristically shaped neurons which are: Responsible for the transmission of electrical impulses and supporting cells, such as glial cells.  3 Neurons are highly differentiated and have not been observed to divide in culture, it needs to nerve growth factor to cultures, the characteristics of nerve cells can be observed with neuroblastomas which are tumor cells that undergo cell growth in culture. 5- Blood and lymph contain a range of cells in suspension. Some of these will continue growth in a culture suspension. These include the lymphoblasts which are white blood cells and are used extensively in culture because" of their ability to secrete immunoregulating compounds" Figure 2.2 Cell types commonly used in culture 3-Selection of particular cell type (How the selection done)? The primary culture will almost certainly contain a variety of different cell types with differing growth capacities. Q//How the experimental work concerned to isolate a single cell type from the culture population?? Answer: There are several ways this can be achieved by the following: Allow the cells to grow. Fast-growing cell types may assume dominance in a population. For example, fibroblasts have relatively short population doubling times and may outgrow other cells after a few generations (called ‘fibroblast overgrowth'). Control the composition of the growth medium. The addition of specific growth factors or known growth inhibitors may allow selective growth of certain cell types. Separate cells by using gradient centrifugation. The cells sediment to an equilibrium position equivalent to their own density—a process called isopycnic sedimentation. The gradients can be formed by nontoxic, high-molecular-weight material such as the formulations ‘Ficoll’ and ‘Percoll’. This method is particularly effective for the isolation of lymphocytes from blood in sterile medium. Cell separation by a simple gradient centrifugation process is shown in Figure below  4 Figure 2.4 Separation of cells by density gradient centrifugation 4- Cells types in culturing? 1- ‘Normal’ cell / / What is a ‘normal’ cell? ‘Normal’ mammalian cells were required as hosts for the production of human vaccines in order to ensure the safety of these products. ‘Normal’ animal cells were defined by Hayflick and Moorhead 1n 1960 with human embryonic cells by a number of characteristics are: a diploid chromosome number (e.g.: 46 chromosomes for human cells). This indicates that no gross chromosomal damage has occurred Anchorage dependence. The cells require a solid substratum for attachment and growth. Growth continues until a confluent monolayer of cells is formed on the substratum. a finite lifespan. This is a reflection of the intrinsic growth potential of the cells; Nonmalignant. The cells are not cancerous. This can be shown by the inability of the cells to form a tumor following injection into immuno-compromised mice. ❖ Anchorage-dependence Q// (What do you means in Anchorage-dependence?) Anchorage-dependence is the requirement of cells for a solid substratum for attachment before growth can occur. At the laboratory scale this substratum can be provided by the solid surface of Petri dishes, T-flasks, or Roux bottles which are made of specially treated glass or plastic. The interaction between the cell membrane and the growth surface is critical and involves a combination of electrostatic attraction and van der Waal’s forces. A negative charge is provided on glass surface containers by alkali treatment. Tissue culture-grade plastic ware consists of sulfonated polystyrene with a surface charge of 2–5 negatively charged groups per nm2.  5 ❖ Cell adhesion occurs by divalent cations (usually Ca2+) and basic proteins forming a layer between the solid substratum and the cell surface. In most cases the cell-surface interaction is provided by a range of nonspecific proteins which form a 2.5 nm-thick layer on the substratum prior to cell attachment. The process of cell attraction to the substratum and the involvement of various proteins in cell-surface bonding represented in: 1-Serum-derived glycoproteins (fibronectin) provide a surface coating conducive to cell attachment. 2- Conditioning factors are released by cells into the medium 3- a bond forming between cell surface glycoproteins and the substratum. ❖ The culture of differentiated cells Differentiation is a process whereby cells slowly change their characteristics to become specialized cells with characteristic phenotypes. This process occurs in vivo during embryo development or during wound healing and leads to the formation of cells with specialized function (differentiated) such as neurons or muscle cells. Differentiation is also associated with normal cell replacement, as is necessary in the bloodstream. The undifferentiated precursors of this process are called stem cells. Most stem cells or embryonic cells grow well in culture. However, as cells 1-become more specialized (differentiated) they tend to lose their growth capabilities 2-this is reflected by poor growth in culture. For most cell types proliferation is incompatible with the expression of differentiated properties. 3- when some cells derived from a tissue are placed in culture there can be an apparent loss in differentiated properties in the surviving cell population.  6 Tumor cells are, in most cases, undifferentiated and have good growth characteristics. However, there are also some differentiated tumor cells which have proved extremely valuable. Differentiated tumor cells retain the phenotypic characteristics of normal differentiated cells but are also able to grow in culture. The following factors may allow some differentiated properties of normal cells to be maintained in culture are: Hormones and growth factors added to media formulations containing selective components that can maintain the differentiated state of specific cell types for example keratinocytes, hepatocytes. Chemical agents. Solvents such as dimethyl sulfoxide (DMSO) may allow the maintenance of a differentiated state by an effect on membrane fluidity. Cell interactions. Contact between cells may allow the formation of gap junctions and allow metabolites to synchronize the expression of differentiation within a cell population. This may also play a part in the arrest of growth when a cell population has covered an available growth surface (defined as ‘confluence'). Interaction with the growth surface. Collagen has been found to be essential for maintaining the polarity of hepatocytes in relation to the attachment surface. Cell polarity is governed by an asymmetrical distribution of ion currents (particularly DMSO). 2- Embryonic stem cells These cells are capable of apparently unlimited growth but have the capacity, given the appropriate stimuli, to differentiate into any other cell type. Human embryonic stem cells were first isolated in 1998 by J. Thomson who derived several cell lines. The cells were derived from the inner cell mass (~30 cells) of a human blastocyte formed from several days' growth of an embryo following in vitro fertilization. These embryonic stem cells have been shown to have several important properties represented in:  7 1- Pluripotent. They have the capacity for differentiation into the cells of the three major tissue types (endoderm, mesoderm and ectoderm). This means that they have the potential to act as precursors for all cells of the body. 2- The cells have a high activity level of telomerase. This tends to correlate with immortality in human cell lines 3- They have a normal diploid karyotype. 4- They can be propagated indefinitely in a non-differentiated state. 5- Directed differentiation. They can be induced to follow a specific pathway of differentiation, given the appropriate chemicals, growth factors or cell contact. 6- They are associated with specific cell markers, e.g. Oct-4 transcription factor and stage specific embryonic antigen (SSEA). ❖ Directed differentiation If embryonic stem cells are allowed to clump, then they form an embryoid body in which the cells begin to differentiate spontaneously. However, through directed differentiation the addition of specific growth factors may direct the cells down a specific pathway of change. 3- Adult stem cells These are undifferentiated cells found among differentiated cells in a tissue or organ. Normally these cells can differentiate along a more limited pathway than embryonic stem cells to produce cells associated with the tissue. The cells(undifferentiated) serve to replace cells or repair tissue damage. Under certain conditions these cells may be induced into cell types other than those associated with the tissue from which they were derived. This is known as transdifferentiation or plasticity and is presently an active area of research. Stem cells found in the bone marrow differentiate through the hematopoietic pathway to provide the extensive range of mature cell types. The hematopoietic stem cells are important for the continuous replacement of the cells found in the blood system. The hematopoietic pathway involves differentiation of cells through four stages—: stem cells, early progenitor cells, progenitor cells and mature cells. The two distinct progenitor lineages (lymphoid and myeloid lineage) are differentiation from hematopoietic system. The lymphoid progenitor cells produce the mature T-lymphocytes, B- lymphocytes and natural killer cells from progenitor cells that are stimulated to differentiate by various interleukins (IL- 2, IL-3, IL-6, IL-7).  8 The myeloid lineage forms erythroid cells that can differentiate into monocytes, macrophages, neutrophils, eosinophils, basophils, megakaryocytes and erythrocytes. The formation of erythrocytes is stimulated by the glycoprotein, erythropoietin "EPO" the production of which occurs in the kidney and is enhanced by low oxygen levels (hypoxia)". The steps of hematopoiesis are controlled by the microenvironment of the cells and secreted proteins (cytokines) from neighboring cells (stromal cells) promote the differentiation process in vivo. Many culture parameters affect the differentiation process including: pH, Osmolarity, temperature and media composition. Figure 2.7 Differentiation of cells of the hematopoietic system. Modified from Inoue, et al., 1995. 4-Transformed cells Transformation has two different meanings in cell biology represented in: Expression of foreign genes in bacteria Change of animal cells from normal to infinite growth capacity. ‘Normal’ animal cells have a finite growth capacity but some cells acquire a capacity for infinite growth and such a population can be called an ‘established’ or ‘continuous’ cell line.  9 Transformed cells is characterized by: may lose their sensitivity to the stimuli associated with growth control. loses their anchorage-dependence and often show some chromosome fragmentation. This genetic state is referred to as aneuploidy, which means that there is a slight alteration from the normal diploid state. The transformed cells have a high capacity for growth in relatively simple growth medium and without the need for growth factors. Carcinogenesis in vivo is analogous, but not identical to the transformation of cells in vitro. Not all transformed cells are malignant, a characteristic defined by the ability to form tumors in animals. However, all tumor-derived cells grow continuously in culture. Examples include: HeLa cells, which are derived from a cervical cancer (Gay et al., 1952). Namalwa cells, which derive from a human lymphoma. These cells are relatively easy to grow and show good growth characteristics, which include a short doubling time and a low requirement for growth factors. ❖ Cells can be transformed or ‘immortalized’ by a variety of techniques. This includes: treatment with mutagens, viruses or oncogenes. An oncogene is defined as a gene that induces the formation of tumorigenic cells. The first ‘tumor’ virus to be recognized was Rous sarcoma virus which is a chicken retrovirus described by Rous in 1911.The viral oncogenes (e.g. v-myc) are derived from cellular —the proto-oncogenes (e.g.: c-myc). Infection by retroviruses is a particularly effective method of immortalizing cells. These retroviruses express activated oncogenes (e.g. myc and ras), which cause cell transformation. The retroviruses are also useful for incorporating recombinant DNA into animal cells ◼ Cells from a culture collection For many applications, cell lines may be obtained from cell culture collections (′cell banks'), which have a large selection of well-characterized cell lines. This is far easier than having to rely on primary animal tissue for establishing cultures. The largest and most well-known international animal cell culture collections are given below. These collections contain well over 3000 well-characterized cell lines. The American Type Culture Collection (ATCC), The European Collection of Animal Cell Culture (ECACC), ◼ Public Health Laboratory Service (PHLS), ◼ Centre for Applied Microbiology Research (CAMR). Prof. Dr. Mahfoodha A. Umran 31 October 2022  Fifth Lecture Animal tissue culture 2022 --- 2023 4th Class 1-SUBCULTURE AND PROPAGATION The first subculture represents an important transition for a culture. When we need to subculture? The need to subculture implies that: The primary culture has increased to occupy all of the available substrate. Cell proliferation has become an important feature. Although the primary culture could have a variable growth fraction after the first subculture depending on the types of cells present in the culture, the growth fraction is usually high (80% or more). Primary culture has a very heterogeneous, containing many of the cell types present in the original tissue, a more homogeneous cell line emerges (appears). This process (subculture) has considerable practical importance, as the culture: Can now be propagated, characterized, and stored, and The potential increase in cell number and The uniformity of the cells opens up a much wide range of experimental possibilities. 2- Cross-contamination and Misidentification There are a number of less desirable (needed or required) consequences of generating a cell line. While propagation and cryopreservation extends the lifetime of a culture and its availability. Who the risk of cross-contamination increases and extends the lifetime of a culture?? 1-Whenever more than one cell line is maintained in a laboratory, there is always a risk that cells from one cell line will be accidentally introduced into the other 2-If its growth rate is faster, it will overgrow and eventually replace the original cell line. The main Causes of cross-contamination in laboratories? Such contamination could be due to: 1- Poor pipetting techniques 2- Sharing media and 3- Pipettes among cell lines, or 4- The generation of aerosols when flasks or media bottles from more than one cell line are open simultaneously. Contamination accidents may also occur at subculture or during cryopreservation from mislabeling, seeding the wrong flask, or poor inventory (register) control in the freezer leading to a cell line becoming misidentified. Example and origin of cross-contamination in cell culture 1  The history of the cell line HeLa makes an interesting story, particularly its origin and biology, and social, and legal implications of its widespread use. when it was found that the majority of continuous cell lines in current use in the 1960s and 1970s had been contaminated with HeLa cells. Cross-contamination or its absence may be confirmed by: 1-Karyotype analysis: chromosome analysis when it was found that the majority of continuous cell lines in current use in the 1960s and 1970s had been contaminated with HeLa cells. 2- Isoenzyme analysis, depending particularly on enzyme polymorphisms wide-scale DNA profiling by cell banks and independent laboratories 3- DNA STR profiling, using standardized STR profiling, an international database of DNA profiles has been created listing cell lines Who the operator working in cell culture avoid cross- contamination? The following practices help avoid cross-contamination: (1) Obtain cell lines from a reputable cell bank (Cell Banks) that has performed the appropriate validation of the cell line," will help prevent accidental importation of infected cell lines. or perform the necessary authentication preferably by DNA STR profiling" (2) Do not have culture flasks of more than one cell line, or media bottles used with them, open simultaneously. (3) Handle rapidly growing lines, such as HeLa, on their own and after other culture (4) Never use the same pipette for different cell lines. (5) Never use the same bottle of medium, trypsin, or other substances for different cell lines. (6) Do not put a pipette back into a bottle of medium, trypsin, or other substances, after it has been in a culture flask containing cells. (7) Add medium and any other reagents to the flask first, and then add the cells last. (8) Check the characteristics of the culture regularly, and suspect any sudden change in morphology, growth rate, or other phenotypic properties. What is the other contamination occurring in cell culture? Mycoplasma Contamination The second major negative consequence of propagating cell lines, and particularly continuous cell lines, is the harboring (hiding) of cryptic contaminations. 1- Most often mycoplasma While infection of a primary culture or an early passage cell line often leads to rapid degeneration and loss of the culture, infection of continuous cell lines seems to be better tolerated and often goes undetected. 2-The main sources of infection are from: 2  a- the operator, b-infected cell lines or tissues, or c- natural substances like serum or trypsin. 3- Terminology " ‫"ا‬ 1-Cell line, once a primary culture is subcultured (or passaged), it becomes known as a cell line. This term implies the presence of several cell lineages of either similar or distinct phenotypes. 2-Cell strain, If one cell lineage is selected, by cloning by physical cell separation, or by any other selection technique, to have certain specific properties that have been identified in the bulk of the cells in the culture, this cell line becomes known as a cell strain. 3-Continuous cell line, if a cell line transforms in vitro, it gives rise to a continuous cell line, and 4- if selected or cloned and characterized, it is known as a continuous cell strain. It is vital at this stage to confirm the identity of the cell lines and exclude (eliminate) the possibility of cross-contamination. How the subculture done in cell culture? & Nomenclature of cell line The first subculture gives rise to a secondary culture, the secondary lead to a tertiary, and so on, although, in practice, this nomenclature is seldom used beyond the tertiary culture. It takes Name of cell line, New cell lines should be given a code name or designation, for example, normal human brain—NHB; a- If several cell lines were derived from the same source, a cell strain or cell line number (then NHB1, NHB2, etc.); b- If cloned, a clone number (NHB2-1, NHB2-2, etc.). It is useful to keep a logbook or computer database file where the receipt of biopsies or specimens is recorded before initiation of a culture c- Sometimes the cell line designation with a code indicating the laboratory in which it was derived (e.g., WI for Wistar Institute, NCI for National Cancer Institute, SK for Sloan-Kettering) Hayflick’s limits The passage number is the number of times that the culture has been subculture, whereas the generation number is the number of doublings that the cell population has undergone, given that the number of doublings in the primary culture is very approximate. 1-When the split ratio is 1:2? What is the split ratio? 3  Hayflick’s work and others with human diploid fibroblasts each subculture divided the culture in half (i.e., the split ratio was 1:2), as in Hayflick’s experiments, the passage number is approximately equal (the same) to the generation number. 2- However, if subculture is performed at split ratios greater than 1:2, the generation number, which is the significant indicator of culture age, will increase faster than the passage number based on the number of doublings that the cell population has undergone since the previous subculture. 4- Culture Age Cell lines with limited culture life spans are known as finite cell lines and behave in a fairly reproducible fashion. Their properties are: 1-They grow through a limited number of cell generations, usually between 20 and 80 cell population doublings, before senescence. 2- The actual number of doublings depends on: Species Cell lineage differences, Conal variation, and Culture conditions, but it is consistent for one cell line grown under the same conditions. 3- a cell line should express the approximate generation number or number of population doublings since explanation, which will be approximate because the number of generations that have elapsed (gone) in the primary culture is difficult to assess. Continuous cell lines have: Escaped from senescence control, The generation number becomes less important and is usually replaced with the number of passages since last thawed from storage becomes more important. In addition, because of the increased cell proliferation rate and saturation density, split ratios become much greater (1:20–1:100) and cell concentration at subculture becomes much more critical. 5- CHOOSING A CELL LINE Apart from specific functional requirements, there are a number of general parameters to consider in selecting a cell line: (1) Finite or continuous. Is there a continuous cell line that expresses the right functions? A continuous cell line generally is easier to maintain, grows faster, clones 4 more easily, produces a higher cell yield per flask, and is more readily adapted to serum-free medium. (2) Normal or transformed. Is it important whether the line is malignantly transformed or not? If it is, then it might be possible to obtain an immortal line that is not tumorigenic, such as 3T3-Swiss cells or BHK21-C13. (3) Species. Is species important? A nonhuman cell line will have fewer biohazard restrictions and have the advantage that the tissue from which it was derived may be more accessible. (4) Growth characteristics. What do you require in terms of growth rate, yield, plating efficiency, and ease of harvesting? To evaluate the Growth characteristics, it will need to consider the following parameters: (a) Population-doubling time (b) Saturation density—yield per flask (c) Plating efficiency. (d) Growth fraction. (e) Ability to grow in suspension). (5) Phenotypic expression. Can the line be made to express the right characteristics? (6) Control cell line. If you are using a mutant, transfected, transformed, or otherwise abnormal cell line, is there a normal equivalent available, are required? (7) Stability. How stable is the cell line? Has it been cloned? If not, can you clone it, and how long would this cloning process take to generate sufficient frozen and usable stocks? Prof. Dr. Mahfoodha Abbas Umran 13 Nov. 2022 5  Sixth Lecture Animal tissue culture 2022 --- 2023 4th Class 6- ROUTINE MAINTENANCE Once a culture is initiated, whether it is a primary culture or a subculture of a cell line, it will need a periodic medium change (feeding or refreshing) followed eventually by subculture if the cells are proliferating. In non-proliferating cultures, the medium will still need to be changed periodically, as the cells will still metabolize and some constituents of the medium will become exhausted (Medium deficiencies can also initiate apoptosis) or will degrade spontaneously. Changing medium Intervals between medium changes and between subcultures vary from one cell line to another, this variation depending on: a- the rate of growth and b- metabolism. 1- Rapidly growing transformed cell lines, such as HeLa, are usually subculture once per week, and the medium should be changed four days later. 2- More slowly growing, particularly nontransformed, cell lines may need to be sub cultured only every two, three, or even four weeks, and the medium should be changed weekly between subcultures. ❖ How could the operator have examined the cell culture? Cell culture be examined carefully to check the status and confirm the absence of contamination. The cells should also be checked firstly by Cell Morphology for any signs of deterioration, such as: 1- granularity around the nucleus, 2-cytoplasmic vacuolation, and 3- rounding up of the cells with detachment from the substrate. Such signs may imply that the culture requires a medium change, or may indicate a more serious problem such as: o inadequate or toxic medium or serum, o microbial contamination, or o senescence of the cell line. Familiarity (understanding) with the cell’s morphology may also allow you to spot the first sign of cross-contamination or misidentification. It is useful to have a series of photographs of cell types in regular use, taken at different cell densities 1 (preferably defined cell densities, e.g., by counting the number of cells/cm2) to refer to when handling cultures. ❖ How the cell culture appears needing to Replacement of Medium? Four factors indicate the need for the replacement of culture medium: (1) A drop in pH. The rate of fall and absolute level should be considered. Most cells stop growing as the pH falls from pH 7.0 to pH 6.5 and start to lose viability between pH 6.5 and pH 6.0, so if the medium goes from red through orange to yellow, the medium should be changed (2) Cell concentration. Cultures at a high cell concentration exhaust the medium faster than those at a low concentration. (3) Cell type. Normal cells (e.g., diploid fibroblasts) usually stop dividing at a high cell density, because of cell crowding, shape change, growth factor depletion. The cells block in the G1 phase of the cell cycle and deteriorate very little, even if left for two to three weeks or longer. Transformed cells, continuous cell lines, and some embryonic cells, however, deteriorate rapidly at high cell densities unless the medium is changed daily or they are sub cultured. (4) Morphological deterioration. This factor must be anticipated by regular examination and familiarity with the cell line. If deterioration (weakening or decline) is allowed to progress too far, it will be irreversible, as the cells will tend to enter apoptosis. 2  6-1 SUBCULTURE& Standard pattern of growth curve When a cell line is sub cultured, there growth of the cells to a point ready for the next subculture usually follows a standard pattern of growth curve represented by: 1-A lag period after seeding more than 24 hours, then it is followed by a period of log phase 2- Exponential growth, called the log phase. It takes from 2 to 7 days or more sometimes depending on cell types. The cell concentration increased logarithmic (exponentially). When the cell density (cells/cm2 substrate) reaches a level such that all of the available substrate is occupied (exhausted), or when the cell concentration (cells/mL medium) exceeds (tops) the capacity of the medium, when it reached to maximum the culture needing subculture, if it is not done, the firstly described at…. 3- Plateau phase, growth ceases or is greatly reduced. Then either the medium must be changed more frequently or the culture must be divided. These phases illustrated in figure below in growth curve and maintenance. How subculture done? For an adherent cell line, dividing a culture, or subculture as it is called, usually involves: removal of the medium and dissociation of the cells in the monolayer with trypsin, although some loosely adherent cells (e.g., HeLa-S3) may be subculture by shaking the bottle, collecting the cells in the medium, and diluting as appropriate in fresh medium in new bottles. Some exceptional cell monolayers cannot be dissociated in trypsin and require the action of alternative proteases, such as 3  Pronase, Dispase (most effective but can be harmful to some cells) and collagenase. Dispase and collagenase are generally less toxic than trypsin but may not give complete dissociation of epithelial cells. The severity of the treatment required depends on the cell type, as does: The sensitivity of the cells to proteolysis, and a protocol should be selected with the least severity with the generation of a single-cell suspension of high viability. With human embryonal stems cells, enzymatic dispersal can lead to the loss of stemness. Subculture must be done mechanically by subdividing a colony of cells and subculturing the pieces. The attachment of cells to each other and to the culture substrate is mediated by: Cell surface glycoproteins and Ca2+. Other proteins, and proteoglycans, derived from the cells and from the serum, become associated with the cell surface and the surface of the substrate and facilitate cell adhesion. Subculture usually requires ❖ chelation of Ca2+ and ❖ degradation of extracellular matrix and, ❖ potentially, the extracellular domains of some cell adhesion molecules. Ideally, a cell concentration should be found that allows for the cells to be sub cultured after 7 days, with the medium being changed after 4 days. 6-2 Criteria for Subculture The need to subculture a monolayer is determined by the following criteria: (1) Density of culture.it differs according to cell types: a- Normal cells should be subcultured as soon as they reach confluence. If left more than 24 h beyond this point, they will withdraw from the cycle and take longer to recover when reseeded. b-Transformed cells should also be subcultured on reaching confluence or shortly after; although they will continue to proliferate beyond confluence. c- Some epithelial cell lines, such as Caco-2, need to be subcultured before they reach confluence as they become too difficult to trypsinize after confluence. 4  (2) Exhaustion of medium. Exhaustion of the medium usually indicates that the medium requires replacement, but if a fall in pH occurs so rapidly that the medium must be changed more frequently, then subculture may be required. Usually a drop in pH is accompanied by an increase in cell density, which is the prime indicator of the need to subculture. (3) Time since last subculture. Routine subculture is best performed according to a strict schedule(timetable), so that reproducible behavior is achieved and monitored. If cells have not reached a high enough density (i.e., they are not confluent) by the appropriate time, then increase the seeding density, or if they reach confluence too soon, then reduce the seeding density. Determination of the correct seeding density and subculture interval is best done by performing a growth curve. (4) Requirements for other procedures. When cells are required for purposes other than routine propagation, they also have to be subcultured in order to increase the stock or to change the type of culture vessel or medium. Ideally this procedure is done at the regular subculture time, when it is known that the culture is performing routinely, what the reseeding conditions are, and what outcome can be expected. Prof..Dr. Mahfoodha Abbas Umran 27 Nov. 2022 5  Seventh lecture Animal Tissue Culture 2021…. 2022 / 4th Class Quantitation Quantitation in cell culture is required for: 1- The characterization of the growth properties of different cell lines, 2- For experimental analyses 3- To establish reproducible culture conditions for the consistency of primary culture and the maintenance of cell line. Cell counting technology and a number of other assays used in quantifying cell proliferation, as well as other basic assays for determining cell bulk, such as DNA and protein estimations. Quantitation by number of cells There are a number of ways of determining cell number, A// the direct, such as  hemocytometer,  electronic counters,  flow cytometry, B// and indirect, such as:  staining with Crystal Violet or  metabolically reduced MTT. 20.1 CELL COUNTING Although cell counting estimates can be made of the stage of growth of a culture from its appearance under the microscope standardization of culture conditions and proper quantitative experiments are difficult to analyze and reproduce unless the cells are counted before and after, and preferably during, each experiment. 20.1.1 Hemocytometer The concentration of a cell suspension may be determined by placing the cells in an optically flat chamber under a microscope (Fig. 20.1). The cell number within a defined area of known depth (i.e., within a defined volume) is counted, and the cell concentration is derived from the count. where c is the cell concentration (cells/mL), n is the number of cells counted, and v is the volume counted (mL). For the Improved Neubauer slide, the depth of the chamber is 0.1 mm, and, assuming that only and all of the central 1 mm2 is used, v is 0.1 mm3, or 1 × 10−4 mL. The formula then namely multiplies your count by 10,000. Becomes: c = n /10−4 or c = n × 104 1  Analysis. Calculate the average of the two counts, and derive the concentration of your sample using the formula: c = n/ v If the cell concentration was high and only the five diagonal squares within the central 1 mm2 were counted (i.e., 1/5 of the total), this equation becomes c = n × 5 × 104 Hemocytometer counting is cheap and gives you the opportunity to see what you are counting. If the cells were previously mixed with an equal volume of a viability stain a viability determination may be performed at the same time. Most of the errors in this procedure occur by: incorrect sampling transfer of cells to the chamber. For this reason, this procedure must be done under this conditions:  Make sure that the cell suspension is properly mixed before you take a sample, and  do not allow the cells time to settle or adhere in the tip of the pipette before transferring them to the chamber.  Ensure also that you have a single-cell suspension, as aggregates make counting inaccurate. 2  Larger aggregates may enter the chamber more slowly or not at all. If aggregation cannot be eliminated during preparation of the cell suspension (see Table 12.5), lyse the cells in 0.1 M citric acid containing 0.1% crystal violet at 37◦C for 1 h and then count the nuclei. 20.1.2 Electronic Counting There are now three main types: (a) The original resistance-based counters (e.g., Beckman Innovatis CASY) based on the change in current generated when a cell passes through a narrow orifice (hole or cavity). Electronic cell counting is rapid and has a low inherent error because of the high number of cells counted. Although resistance counting is prone to misinterpretation, because cell aggregates, dead cells, and particles of debris of the correct size will all be counted, corrections are possible to exclude dead cells and aggregates and the CASY can make an approximate programmatic correction for aggregation. (b) Image analysis of a microscope view of unstained or stained cells or nuclei in special counting chambers by visible light or fluorescence (e.g., Invitrogen Countess, Peqlab Cellometer or Chemometic Nucleopcounter), and Cell counting by image analysis. Image analysis is used to scan cells in an optical counting chamber and will discriminate between live and dead cells by recognizing Trypan Blue staining (e.g., Coulter Vi-CELL) or, in some models, fluorescence from propidium iodide (PI; e.g.,Digital- Bio ADAM). This can be viewed on the display. Most of these counters can also generate an analog plot of cell size distribution, enabling upper and lower thresholds to be set. A sample of cells, usually 20 μL to 1 mL, is injected into the chamber and placed in the counter. The range of cell concentrations that can be used range from 5 × 104 to 1 × 107 cells/mL for those using Trypan Blue, while those using PI can be used down to 5 × 103 cells/mL. After setting the correct thresholds, the count takes about 30 s. Counters using PI (e.g., Nucleocounter) can also estimate viability from the ratio of PI uptake in untreated cells divided by total PI uptake in lysed cells in the same sample. (c) Bench-top flow cytometers that analyze a single cell stream for cell concentration and other parameters (e.g., Guava, Accuri). 20.1.3 Stained Monolayers There are occasions when cells cannot be harvested for counting or are too few to count in suspension (e.g., at low cell concentrations in microtitration plates). In these cases the cells may be fixed and stained in situ and counted by eye with a microscope , although these measurements may not correlate directly with the cell number—for example, if the ploidy of the cell varies. A rough estimate of the cell number per well can also be obtained by staining the cells with Crystal Violet and measuring the absorption on a densitometer. This method has also been used to calculate the number of cells per colony in clonal growth assays.  Staining cells with Coomassie Blue, sulforhodamine B , or  MTT. MTT staining has the advantage that it stains only viable cells. 3  Fig. 20.6. Accuri C6 Flow Cytometer. Upper panel, cytometer and associated PC. Lower panel,typical screen output. (Courtesy of Accuri Cytometers.) 20.1.4 Flow Cytometry Flow cytometry of a cell suspension [Shapiro, 2003; Applied Cytometry, 2008], while losing the relationship between cytochemistry and morphology, samples up to 1 × 107 cells.The main properties can measure multiple cellular constituents and activities and it enables correlation of these measurements with other cellular parameters, such as cell size, lineage, DNA content, or viability. While multiparametric analysis and cell sorting are best performed with one of the more elaborate machines, such as the FACStar, small bench-top flow cytometers can give useful information about the status of the cells in addition cellular material. DNA may be assayed by several fluorescence …. to a cell count and are not much more expensive than an electronic cell counter (Fig. 20.6). Parameters such as viability (by propidium iodide uptake) or apoptosis (by annexin V immunostaining) can be readily quantified (Fig. 20.7). 4  Fig. 20.7. Output from Guava Flow Cytometer. Dot plot from deteriorating cell culture. (a–d) Four panels show progressive accumulation of dead cells, stained with PI, in upper right quadrant. (e) Bottom two panels show apoptotic cells, staining with antibody to Annexin V (bottom right quadrant in right-hand image). (Courtesy of Edward Burnett, ECACC.) 20.2 '' CELL WEIGHT'' A // Wet weight is seldom used unless very large cell numbers are involved because the amount of adherent extracellular liquid gives a large error. As a rough guide, there are:  about 2.5 × 108 HeLa cells (14–16 μm in diameter) per gram wet weight,  about 8 to 10 × 108 cells /g for murine leukemias, such as L5178Y murine lymphoma, myelomas, and hybridomas (11–12 μm in diameter),  and about 1.8 × 108 cells/g for human diploid fibroblasts (16–18 μm m in diameter). B // dry weight is seldom used because  salt derived from the medium contributes to the weight of unfixed cells, and  fixed cells lose some of their low-molecular-weight intracellular constituents and lipids. 20.3 ''DNA CONTENT'' In practice, the cell number, DNA and protein are the two most useful measurements for quantifying the amount of methods, including reaction with  DAPI, PicoGreen (assay kit from Molecular Probes), or  Hoechstm33258. The fluorescence emission of Hoechst 33258 at 458 nm is increased by interaction of the dye with DNA at pH 7.4 and in high salt to dissociate the chromatin protein. This method gives a sensitivity of 10 ng/mL but requires intact double-stranded DNA. DNA can also be measured by its absorbance at 260 nm, where 50 μg/mL has an optical density (O.D.) of 1.0. Because of interference from other cellular constituents, the direct absorbance method is useful only for purified DNA. 5  20.4 '' PROTEIN'' The protein content of cells is widely used for estimating total cellular material and can be used in  1- growth experiments or as a denominator in expressions of the specific activity of enzymes,  2- the receptor content, or  3- intracellular metabolite concentrations. The amount of protein in solubilized cells can be estimated directly by measuring the absorbance at 280 nm, with minimal interference from nucleic acids and other constituents. The absorbance at 280 nm can detect down to 100 μg of protein, or about 2 × 105 cells. Colorimetric assays are more sensitive than measurements of UV absorption, and among these assays, the Bradford reaction with Coomassie Blue [Bradford, 1976] is one of the most widely used. 20.4.1 Solubilization of Sample Because most assays rely on a final colorimetric step, they must be carried out on clear solutions. Cell monolayers and cell pellets may be dissolved in 0.5 to 1.0 M NaOH by heating them to 100◦C for 30 min or leaving them overnight at room temperature. Alternatively, with 0.3 M NaOH and 1% sodium lauryl sulfate, the solution is complete after 30 min at room temperature. 20.4.2 Bradford Assay The Bradford method is not dependent on specific amino acids and is quite sensitive, requiring 50 to 100,000 cells. Coomassie Blue undergoes a spectral change on binding to protein in acidic solution. Color is generated in one step after a short incubation and should be read within 30 min. 20.5 ''RATES OF SYNTHESIS'' 20.5.1 DNA Synthesis Measurements of DNA synthesis are often taken to be representative of the amount of cell proliferation. [3H]thymidine ([3H]-TdR) or [3H]deoxycytidine is the usual precursor that is employed. Exposure to one of these precursors may be for short periods (0.5–1 h) for rate estimations or for longer periods (24 h or more) to measure accumulated DNA synthesis when the basal rate is low (e.g., in high-density cultures).  [3H]-TdR should not be used for incubations longer than 24 h or at high specific activities as radiolysis of DNA will occur might releases energy within the nucleus and causes DNA strand breaks  [14C]-TdR or 32P should be used, If prolonged incubations or high specific activities are required. 20.5.2 Protein Synthesis Colorimetric assays measure the total amount of protein present at any one time. Sequential observations over a period of time may be used to measure the net protein accumulation or loss (i.e., protein synthesized–protein degraded), while the rate of protein synthesis may be determined by incubating cells with a radioisotopically labeled amino acid, such as [3H] leucine or [35S] methionine, and measuring (e.g., by scintillation counting) the amount of radioactivity incorporated into acid- insoluble material per 1 × 106 cells or per milligram of protein over a set period of time. ) ‫ الجزء الثاني للمحاضرة سيكون هو (المحاضرة الثامنة‬/ ‫الى هنا الجزء االول للمحاضرة السابعة‬ Prof..Dr..Mahfoodha Abbas Umran 3 Dec. 2021 6  Eighth lecture/ B Animal Tissue Culture 4th Class/ Dec. 2022 Quantitation / Part B 20.6 ''PREPARATION OF SAMPLES FOR ENZYME ASSAY AND IMMUNOASSAY'' As the amount of cellular material available from cultures is often too small for efficient homogenization, so other methods of lysis are required to release: Soluble products and Enzymes for assay. It is convenient either to a- Set up cultures of the necessary cell number in sample tubes or b- Multi well plates or to trypsinize a bulk culture and place aliquots of cells into assay tubes. ❖ In either case the cells should be lysis as following : 1- washed in HBSS or D-PBSA to remove the serum, and 2- lysis buffer should be added to the cells. The lysis buffer should be chosen to suit the assay, but If the particular lysis buffer is unimportant, 0.15 M NaCl or D-PBSA may be used. If the product to be measured is membrane bound, add 1% detergent (Na deoxycholate, Nonidet P40) to the lysis buffer. ❖ If the cells are pelleted, Resuspend them in the buffer by vortex mixing. Freeze and thaw the preparation three times by placing the sample tube in EtOH containing solid CO2 (∼−90◦C) for 1 min and then in 37◦C water for 2 min (longer for samples greater than 1 mL). Finally, spin the preparation at 10,000 g for 1 min (e.g., in an Eppendorf centrifuge), and Collect the supernatant for assay. Alternatively, the whole extract may be assayed for enzyme activity, and the insoluble material may be removed by centrifugation later if necessary. 20.7 ''CYTOMETRY'' 7.1 In situ Labeling Fluorescence labeling, accomplished either: Directly with a fluorescent dye (e.g., Hoechst 33258 or DAPI for DNA) or In directly with a conjugated antibody for detection of an antigen or molecular probe, can measure the amounts of enzyme, DNA, RNA, protein, or other cellular constituents in situ with a CCD camera. This process allows qualitative and, when image analysis is used, quantitative analyses to be made, but is slow if large numbers of cells are to be scanned. 7.2 Flow Cytometry Fluorescence labeled cell populations can also be quantified by flow cytometry where 1 1-a very precise quantitative analysis may be made but in the absence of any structural relationships among the cells. 2- Additional parameters that can be measured in a flow cytometer include forward and backward light scatter (influenced by cell size and surface configuration) and chromogenic enzyme substrates or products. 3- Although flow cytometers used in cell separation (see Section 14.4) tend to( be likely to ) be large and expensive machines, there are several low-cost bench-top analytical machines available, such as Guava and Accuri. 20.8 ''REPLICATE SAMPLING'' Because in most cases cultured cells can be prepared in a uniform suspension, the provision of large numbers of replicates for statistical analysis is often unnecessary. Usually three replicates are sufficient, and for many simple observations (e.g., cell counts), duplicates may be sufficient. Many types of culture vessel are available for replicate monolayer cultures, and the choice of which vessel to use is determined: (a) by the number of cells required in each sample and by the frequency or type of sampling. For example, Multiwell plates, such as microtitration plates or 24-well plates performed in replicate sampling is most readily when the incubation time is not a variable, Individual tubes or 4-well plates are best prepared in this case the replicates. Samples are collected over a period of time (e.g., daily for 5 days), then the constant removal of a plate for daily processing may impair (damage) growth in the rest of the wells. Plain glass or tissue culture-treated plastic test tubes may be used, although Leighton tubes are superior, as they provide a flat growth surface. Alternatively, if the optical quality of the tubes is not critical, flat-bottomed glass specimen tubes and even glass scintillation vials may be good containers. If glass vials or tubes are used, they must be washed as tissue culture glassware they cannot be used for tissue culture after use with scintillant. How the tubes sealed? Sealing large numbers of vials or tubes can become tedious, so many people seal tubes with Vinyl tape rather than screw caps. Such tape can also be color coded to identify different treatments. Adhesive film may be used for sealing microtitration plates. This reduces evaporation and contamination and gives a more even performance across the plate. It also means that individual wells or rows can be sampled without opening up the rest of the plate. ❖ Handling suspension cultures is generally easier than dealing with monolayer cultures because the shape of the container and its surface charge are less important. ❖ Multiple sampling can also be performed on one culture when using suspension cultures. This sampling is done conveniently by sealing the bottle containing the culture with a silicone rubber membrane closure (Pierce) and then sampling with a syringe and needle. (Remember to replace the volume of culture removed with an equal volume of air.) 20.9 ''CELL PROLIFERATION'' Measurements of cell proliferation rates are often used to : 1-Dterminethe response of cells to a particular stimulus or toxin Quantitation of culture growth is 2--Important in routine maintenance, as It is a crucial element for monitoring the consistency of the culture and 2  3- knowing the best time to subculture, the optimum dilution, and the estimated plating efficiency at different cell densities. Testing medium, serum, new culture vessels or substrates, and so forth, all require quantitative assessment. As with cell counting there are a number of different ways of determining cell proliferation using: ❖ cell counting, ❖ plating efficiency, or ❖ labeling with radioisotopic precursors of DNA or antibodies to cell cycle specific proteins (Table 20.4). Counting and plating may be seen direct, While labeling techniques are indirect as imply proliferation from the expression of metabolic markers. 9.1 Experimental Design Growth state of a culture, and its kinetic parameters, is critical in the design of cell culture experiments. Cultures vary significantly in many of their properties between the lag phase, the period of exponential growth (log phase), and the stationary phase (plateau). It is therefore important to take: account of the status of the culture both at the initiation of an experiment and at the time of sampling, in order to determine whether is it proliferating or not and, the duration of the population doubling time (PDT) and the cell cycle time. In the plateau phase , the Cells that have entered this phase have 1- a greatly reduced growth fraction and 2- a different morphology, may be more differentiated, and may become polarized. 3-They tend to secrete more extracellular matrix and may be more difficult to disaggregate. In the log phase, generally, cell cultures are 1- Consistent and uniform, and 2-Sampling at the end of the log phase gives the highest yield and greatest reproducibility. It is also important to consider the effects of the duration of an experiment on the transition from one state to another. 3- Adding a drug in the middle of the exponential phase and assaying later may give different results, depending on whether the culture is still in exponential growth when it is assayed or whether it has entered the plateau phase. Microtitration plate assays of cytotoxicity are particularly susceptible to error if the culture reaches plateau during an assay; as the cells in those wells that are at the highest density reach plateau, cell proliferation decreases and there is an apparent shift in the 50% inhibitory point (ID50) as more wells reach plateau. 9.2 Growth Cycle After subculture, cells progress through a characteristic growth pattern of lag phase, exponential, or log phase, and stationary, or plateau phase (Fig. 20.8;). 3  ❖ The log and plateau phases give vital information about the cell line, the PDT during exponential growth, and the maximum cell density achieved in the plateau phase (i.e., the saturation density). The measurement of the" PDT" is used to: 1- Quantify the response of the cells to different inhibitory or stimulatory culture conditions, such as variations in nutrient concentration, hormonal effects, or toxic drugs. 2- It is also a good monitor of the culture during serial passage and enables the calculation of cell yields and the dilution factor required at subculture. Single time points are unsatisfactory for monitoring growth if the shape of the growth curve is not known. A reduced cell count after, say, 5 days could be caused by a reduced growth rate of some or all of the cells; ❖ a longer lag period, implying adaptation or cell loss (it is difficult to distinguish between the two); or a reduction in saturation density The importance of "growth curves" is implying to be useful: ❖ for testing media, sera, growth factors, and some drugs, and once the response being monitored is fully characterized and the type of response is predictable (e.g., a change in the PDT), then single time point observations can be made at a time point known to be in mid-log phase. ❖ for the de