Cell Culture And Tissue Culture PDF

Unit 1 General Principles of Biological Analysis Physiological Solution A physiological solution, also known as a physiological saline solution or simply saline, refers to a solution that closely mimics the composition of bodily fluids, such as blood plasma. The primary purpose of physiologic...

Unit 1 General Principles of Biological Analysis Physiological Solution A physiological solution, also known as a physiological saline solution or simply saline, refers to a solution that closely mimics the composition of bodily fluids, such as blood plasma. The primary purpose of physiological solutions is to provide a suitable environment for cells, tissues, and organs to maintain their normal physiological functions. These solutions are isotonic, meaning they have the same osmotic pressure as bodily fluids. Physiological solutions are widely used in various biological and medical applications, such as intravenous (IV) infusions, cell culture, and in laboratory experiments. The composition of a physiological solution typically includes: Sodium chloride (NaCl): This is the main salt component and helps maintain osmotic balance. Potassium chloride (KCl): Contributes to the maintenance of cell membrane potential. Calcium chloride (CaCl2): Important for cell signaling and maintaining cell structure. Sodium bicarbonate (NaHCO3): Helps to regulate the pH of the solution. Water: The solvent for the salts and essential for maintaining hydration. Physiological solutions, also known as saline solutions, come in various types depending on their specific compositions. These solutions are designed to mimic the electrolyte balance and osmolarity of bodily fluids. Normal Saline (0.9% Sodium Chloride): Lactated Ringer's Solution: Hartmann's Solution: Hypertonic Saline: Hypotonic Saline: Dextrose Saline: Buffered Solutions: Normal Saline (0.9% Sodium Chloride): This solution contains 0.9% w/v (weight/volume) sodium chloride (NaCl) dissolved in water. It is isotonic with blood plasma and is often used for intravenous (IV) fluid replacement to restore electrolyte balance. Normal saline is widely used in medical settings for hydration and as a diluent for medications. Hartmann's Solution: Similar to Lactated Ringer's solution, Hartmann's solution is an isotonic electrolyte solution used for fluid resuscitation. It contains sodium chloride, potassium chloride, calcium chloride, and sodium lactate. It is often used in surgical and critical care settings. Hypertonic Saline: Hypertonic saline solutions have a higher concentration of sodium chloride than normal saline. They are used in specific medical situations, such as in the treatment of hyponatremia (low blood sodium levels) or for cerebral edema. The higher osmolarity can draw water out of cells. Dextrose Saline: Dextrose saline solutions combine sodium chloride with dextrose (glucose). These solutions provide both electrolytes and a source of energy (calories). They are often used in IV therapy to provide fluid and nutrients. Cell Culture: Cell culture refers to the process of growing and maintaining cells outside their natural environment, typically in a controlled laboratory setting. This is done in special containers called cell culture dishes or flasks. The cells can be derived from various sources, including animal tissues, plants, or microorganisms. Components of Cell Culture: Culture Medium: This is a nutrient-rich solution that provides the necessary nutrients for cell growth. It includes amino acids, vitamins, salts, glucose, and other essential components. Serum: Fetal bovine serum (FBS) or other types of serum are often added to the culture medium. Serum provides growth factors, hormones, and other factors essential for cell growth. CO2 Incubator: Maintains a controlled environment with temperature, humidity, and carbon dioxide levels to mimic the physiological conditions required for cell growth. Tissue Culture: Tissue culture is an extension of cell culture where not only individual cells but entire tissues are cultured in vitro. Tissues can be derived from various organs and organisms. Applications of Cell and Tissue Culture: Medical Research: Studying cell behavior, disease mechanisms, and drug testing. Biotechnology: Production of therapeutic proteins, vaccines, and other biopharmaceuticals. Toxicology Studies: Evaluating the effects of chemicals and drugs on cells and tissues. Regenerative Medicine: Growing tissues or organs for transplantation.  In both cell and tissue culture, maintaining a physiological environment is crucial for the cells to proliferate, differentiate, and function properly. The use of physiological solutions ensures that the osmotic pressure, pH, and nutrient levels closely resemble the in vivo conditions, promoting successful cell and tissue growth in the laboratory setting.  Forensic significance 1.DNA Analysis: Cell Culture for DNA Profiling: In forensic DNA analysis, the cultivation of cells from biological samples, such as blood or tissue, can be crucial. Cell cultures help in obtaining a larger quantity of DNA for analysis, especially when the initial sample is limited. 2. Biological Fluid Analysis: Preservation of Biological Evidence: Physiological solutions play a role in preserving biological samples found at crime scenes. Saline solutions are often used to prevent degradation of biological materials, maintaining the integrity of cells and tissues for subsequent analysis. 3. Toxicology Studies: Postmortem Tissue Culture: In cases of suspicious deaths or toxic exposure, postmortem tissue cultures may be conducted to understand the effects of toxins or drugs on tissues. This can aid in determining the cause of death or providing evidence of poisoning. 4.Forensic Pathology: Tissue Preservation: Physiological solutions are used in the preservation of tissues obtained during autopsies. This ensures that the tissues maintain their structural and cellular integrity, allowing forensic pathologists to conduct thorough examinations and analyses. 5. Biological Fluid Typing: Cell Culture for Blood Typing: Cell cultures can be used for blood typing and other serological analyses. This can be important in cases where blood evidence is critical in determining the identity of victims or suspects. 6. Forensic Anthropology: Bone Tissue Analysis: In cases involving skeletal remains, cell and tissue culture can be applied to extract DNA from bone tissues. This is particularly useful when traditional DNA extraction methods may not be feasible. 7. Crime Scene Investigation: Preservation of Evidence: Physiological solutions are used to preserve evidence collected from crime scenes, such as biological fluids, tissues, or organs. This preservation is essential for maintaining the viability of cellular materials during transportation to the laboratory. 8. Individual Identification: Cell Line Establishment: In cases where individual identification is challenging, establishing cell lines from biological samples can aid in the long-term preservation of genetic information for future analysis or comparison. Preparation of equipment and Materials Process of Cell Cell Isolation Culture Seeding Cells Incubation Subculturing (passaging) Cryopreservation Thawing cells Monitoring and Maintenance  Process of Cell Culture 1. Preparation of Equipment and Materials Sterilization: Ensure that all equipment (pipettes, flasks, dishes) is sterilized using autoclaving, ethanol, or UV light to prevent contamination. Culture Media Preparation: Prepare the appropriate growth medium containing essential nutrients, salts, amino acids, and growth factors. It should be sterile and adjusted to the correct pH. 2. Cell Isolation Primary Culture:Tissue Disaggregation: For primary cells, tissue is obtained and disaggregated using mechanical methods (mincing) or enzymatic digestion (trypsin, collagenase). Filtration: Pass the cell suspension through a mesh to remove debris. Cell Line Sourcing: Alternatively, an established cell line can be obtained from a repository. 3. Seeding Cells Count the cells using a hemocytometer or an automated cell counter. Dilute the cells in the culture medium to achieve the desired seeding density. Plate the cells into appropriate culture vessels (e.g., Petri dishes, T-flasks). 4. Incubation Place the culture vessel in an incubator set to the optimal conditions for the cell type (usually 37°C, 5% CO₂, and 95% humidity). Check daily for contamination, growth, and morphology changes using a microscope. 5. Subculturing (Passaging)Purpose: Prevent cells from becoming overcrowded and maintain exponential growth. Steps: Remove spent medium. Wash the cells with sterile PBS to remove debris. Add trypsin-EDTA to detach adherent cells from the vessel surface. Neutralize trypsin using complete medium. Centrifuge the suspension to pellet the cells. Resuspend the cells in fresh medium and transfer them to a new culture vessel. 6. Cryopreservation (Optional) Purpose: Store cells for long-term use. Steps: Detach and collect cells as for passaging. Mix cells with a cryoprotectant (e.g., 10% DMSO in freezing medium). Aliquot into cryovials. Gradually freeze the cells to -80°C and then transfer to liquid nitrogen for long-term storage. 7. Thawing Cells (revive) Quickly thaw frozen cells in a water bath at 37°C. Transfer the thawed suspension to pre-warmed culture medium. Centrifuge to remove the cryoprotectant and reseed into a culture vessel. 8. Monitoring and Maintenance Daily Checks: Observe for contamination, pH changes (indicated by color change in phenol red-containing medium), and cell morphology. Media Changes: Replace spent medium regularly to maintain nutrient levels and remove waste products.  https://study.com/academy/lesson/cell-fractionation-definit ion-steps-methods.html https://modernbio.com/blog/what-is-cell-fractionation/ https://gyansanchay.csjmu.ac.in/wp-content/uploads/2021 /11/Cell-Fractionation-Swasti.pdf https://www.slideshare.net/MmeesawMeesaw/fractionatio n-of-cells best Cell Fractionation? Cells are fluid-filled microscopic pouches protected by a lipid bilayer. Inside of eukaryotic cells are miniature organs, called organelles, which each have different functions. If scientists want to study the internal parts of a cell, they have to perform cell fractionation. What is cell fractionation? Cell fractionation is a multistep procedure that subjects cells to centrifugal force in a spinning device called a centrifuge, which separates organelles by their physical properties. The purpose of cell fractionation is to isolate the organelles within a cell so scientists can study them individually.  Cell Fractionation Cell fractionation is a laboratory technique used to separate cellular components based on their size, density, and other properties. This process allows scientists to study and analyze the different organelles and structures within a cell. The following are the general steps involved in cell fractionation: Cell Fractionation Procedure Samples or tissues containing the cells of interest are collected and prepared in isotonic buffered solution at a cold temperature. Some examples of tissues a scientist might be interested in studying are leaves from a plant, a mouse liver, a segment of a tapeworm, or a sample of blood. The cell fractionation procedure involves two steps, which are: Breaking the cells open (via homogenization) Separating and isolating the organelles (via centrifuge)  Cell Culture or Tissue Preparation: If working with cultured cells, they are typically grown to confluence. For tissues, they are harvested and minced into small pieces.  Homogenization: The cell or tissue sample is homogenized to break the cell membrane and release cellular components. This can be done using various methods, such as mechanical homogenization, Dounce homogenization, or using a homogenizer. The choice of homogenization method depends on the type of cells or tissues being processed. A cell homogenate, or cell lysate, is created when the cellular components are mashed together. In truth, detergents and soaps remove oil and grease by allowing substances that do not ordinarily combine with water to dissolve in water and rinsed away. Centrifugation (First Spin - Low Speed): The homogenate is subjected to a low-speed centrifugation (typically around 1,000 to 2,000 x g) to separate the nuclear fraction (pellet) from the cytoplasmic fraction (supernatant). The resulting supernatant, called the post-nuclear supernatant (PNS), contains organelles such as mitochondria, endoplasmic reticulum, and Golgi apparatus.  Centrifugation (Second Spin - Medium Speed): The PNS is further centrifuged at a higher speed (around 10,000 to 20,000 x g). This separates heavier organelles like mitochondria and lysosomes from lighter components like microsomes and cytosol. The pellet obtained may contain a crude mitochondrial fraction, while the supernatant (cytosolic fraction) can be further processed.  Centrifugation (Third Spin - High Speed): The supernatant from the medium-speed centrifugation is subjected to a high-speed centrifugation (around 100,000 x g). This step helps to separate smaller organelles like microsomes (containing endoplasmic reticulum) from the cytosol. The resulting pellet may contain microsomes, while the supernatant is the cytosolic fraction.  Density Gradient Centrifugation (Optional): To further purify specific organelles or membranes, a density gradient centrifugation may be performed. This involves layering the sample on top of a density gradient medium and subjecting it to ultracentrifugation. Different organelles or components migrate to different positions in the gradient based on their density. Isolation and Analysis: The fractions obtained from the centrifugation steps can be collected, and their purity can be assessed using various techniques such as Western blotting, enzyme assays, or electron microscopy. Researchers can then use the isolated organelles or fractions for further studies, such as biochemical assays, proteomic analysis, or functional experiments. Cell culture Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This technique is also called micropropagation. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. They need to be kept at body temperature (37 °C) in an incubator. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or rich medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants. The lifespan of most cells is genetically determined, but some cell-culturing cells have been “transformed” into immortal cells which will reproduce indefinitely if the optimal conditions are provided. the term "cell culture" now refers to the culturing of cells derived from multicellular eukaryotes, especially animal cells