SIO2004 Animal Cell and Tissue Culture Lecture 2 Biotech. Program, University of Malaya PDF

SIO2004 Animal Cell and Tissue Culture Lecture 2 Biotechnology Program University of Malaya Instructor: Dr. Nuradilla Mohamad Fauzi  Let’s start a cell culture! Wait. Where do we get the cells from? How do you isolate cells? Tissue biopsy...

SIO2004 Animal Cell and Tissue Culture Lecture 2 Biotechnology Program University of Malaya Instructor: Dr. Nuradilla Mohamad Fauzi  Let’s start a cell culture! Wait. Where do we get the cells from? How do you isolate cells? Tissue biopsy Initiation of culture Cell cultures may be derived from: Explant culture Dissociated cell culture  A tissue biopsy is taken Explant culture Biopsy is dissected, cut into small pieces Pieces of tissue are placed on add media! the culture surface (explants!) Cells will migrate out from the explant Only adherent cells with ability to migrate Primary culture: 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. Part of the virtual lab sessions will be about establishing this type of culture! Figure. Primary explant culture from human atrial explant and c- kit pos cardiac stem cell (CSC) sorting. Explant ( a ) and early stage outgrowth at 3, 7, and 14 days after explantation ( b – e ). Outgrowth confluent cells away from the explant at 28 days after explantation ( f ). c- kit pos cells were sorted by flow-activated cell sorter ( g ). Single c-kit pos cell suspension and formation of a clone of human c-kit pos CSCs ( h – i ). DOI: 10.1007/s00018-007-6519-y Dissociation/disaggregation A tissue biopsy is taken Mechanical and/or enzymatic dissociation of tissue Mechanical, e.g. serial pipetting, mincing with razor Enzymatic, e.g. trypsin and collagenase add media! Tissue “slurry” is placed on the culture surface You get both adherent and non-adherent cells (that survive the dissociation) Primary culture: Dissociated cell culture Maintenance of growth of cells dissociated/disaggregated from the parental tissue using mechanical or enzymatic methods, in culture medium using suitable glass or plastic containers https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410322/  Animal Plant Tissue Primary culture Subculture heterogeneous Stored Stored H Cell line Continuous cell line  Wenfeng Li, Van Thao Nguyen, Mathias Corteel, João José Dantas-Lima, Khuong Van Thuong, Vo Van Tuan, Peter Bossier, Patrick Sorgeloos, Hans Nauwynck, Characterization of a primary cell culture from lymphoid organ of Litopenaeus vannamei and use for studies on WSSV replication, Aquaculture, Volume 433, 20 September 2014, Pages 157-163, ISSN 0044-8486, http://dx.doi.org/10.1016/j.aquaculture.2 014.05.046. Monolayer of primary cell culture developed from lymphoid organ of L. vannamei. A: round cells migrating from explants at 24 h post seeding; B: fusiform cells with branches transformed from round cells at 48 h post seeding; C: fibroblast-like cells that grew out from explants at 48 h post seeding; D: epitheloid cells at 72 h post seeding (scale bar = 30 μ m) Initiation of culture Cell cultures may be derived from primary explants or dispersed cell suspensions. Tissue samples are invariably heterogeneous. Replicates, even from one tissue, vary in their constituent cell types. In explant culture, you get cells that have the capacity to migrate from the tissue explant. With dissociation/disaggregation, you only get those cells that both survive the disaggregation technique and adhere to the substrate or survive in suspension. Regardless of the method employed, primary culture is the first in a series of selective processes that may ultimately give rise to a relatively uniform cell line. fast Animal Plant medium Tissue slow Primary culture passage Subculture heterogeneous Cell line homogeneous Stored multiple passages Stored H Cell line Continuous cell line  Selection of cell populations A primary culture is a mix of different cell populations (heterogeneous) With repeated passaging in normal culture conditions, cells with high proliferative capacity (divide fast) and robustness will dominate Standard in vitro culture conditions promote proliferation! After repeated passaging, the culture eventually becomes homogeneous and can be called a cell line. In a mixed primary culture, differences in growth rate may mean a loss of the cell type of interest. Therefore, selection techniques need to be used to select for your cell type of interest if it is a slow-growing one! Using specific medium and additives Using specific substrates Adjusting culture conditions! Cell sorting (flow cytometry) We will learn about flow cytometry later in this course!  Animal Plant Tissue Primary culture Subculture Stored Stored Cell line Continuous cell line  From primary culture to cell line The resulting monolayer or cell suspension may be dispersed by enzymatic treatment or simple dilution and subcultured into fresh vessels. This constitutes a subculture or passage. The daughter cultures so formed are the beginnings of a cell line. After a few passages, the cultured cell line will assume a homogeneous (or uniform) constitution 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. Cell line The formation of a cell line from a primary culture implies: (1) an increase in the total number of cells over several generations (population doublings) (2) the ultimate predominance of cells or cell lineages with a high proliferative capacity (3) a degree of uniformity in the cell population A cell line will retain specific characteristics throughout (most) of its lifespan. A cell line may undergo transformation to become a continuous cell line (“immortal”) (we will learn more about transformation later!) But … what if we want the slower-growing cells instead?  Selection of cell populations A primary culture is a mix of different cell populations (heterogeneous) With repeated passaging in normal culture conditions, cells with high proliferative capacity (divide fast) and robustness will dominate Standard in vitro culture conditions promote proliferation! After repeated passaging, the culture eventually becomes homogeneous and can be called a cell line. In a mixed primary culture, differences in growth rate may mean a loss of the cell type of interest Therefore, selection techniques need to be used to select for your cell type of interest if it is a slow-growing one! Using specific medium and additives Using specific substrates Adjusting culture conditions! Cell sorting (flow cytometry) We will learn about flow cytometry later in this course!  Cells may be propagated as an adherent monolayer or in suspension. Monolayer culture signifies that the cells are grown attached to the substrate. Anchorage dependence: attachment to the substrate is a prerequisite for cell proliferation. Monolayer culture is the mode of culture common to most normal cells, with the exception of hematopoietic (blood) 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.  Because they may be propagated as a uniform cell suspension or monolayer, cell cultures have many advantages Quantitation: easy to count cells Characterization: see morphology of cells, visualization of individual cells and structures Easy to make replicate samples (just prepare multiple dishes of cells by passaging!) Vascular smooth muscle cells, isolated from human aorta, growing and forming a monolayer in cell culture.  Adherent Cell vs. Suspension Cell Adherent Cell Culture Suspension Cell Culture Attached to a solid or semi-solid substrate Grown floating in the culture medium Easy visual inspection under inverted Requires daily cell counts and viability microscope determination to follow growth patterns Cells are dissociated enzymatically (e.g. Does not require enzymatic or mechanical trypsin) or mechanically dissociation Growth is limited by surface area Growth is limited by concentration of cells No agitation required Requires agitation (i.e., shaking or stirring) for adequate gas exchange Used for cytology, harvesting products Used for bulk protein production, batch continuously, and many research harvesting, and many research applications applications  What if we want the stem cells from the Wharton’s Jelly?  Critically ill COVID-19 patients treated with non- altered stem cells from umbilical cord connective tissue were >2x as likely to survive as those who did not have the treatment. The trial used stem cells obtained through explants from actual umbilical cord tissue. https://www.eurekalert.org/news-releases/827321 Now that you have isolated cells, what kind of cells do you actually get? Primary culture freshly isolated from tissue source Cell line Finite cell line: dies after several sub-cultures Continuous cell line: ‘immortal’ cancer cells, ‘transformed’ cells Generally, cells that can be passaged and maintained in culture are cells that can divide! (not all cells can) Different cell types can have different morphologies that, in part, corresponds to their functions in vivo. Morphology of cells in culture Most mammalian cells in culture can be divided in to three basic categories based on their morphology. Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate. Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches. Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface. In addition to the basic categories listed above, certain cells display morphological characteristics specific to their specialized role in host. FIG. In vitro morphology of TM (eardrum) explants and cultured cells. (A) Explant culture of a (whole) normal unperforated rat tympanic membrane showed cell outgrowth after 3 days in culture. (B) Physical trauma to the TM (3 day TMP) appears to enhance cell outgrowth at the same time point. (C) Excised umbo showed a delayed cell outgrowth at day 5 in culture. (D) Excised TM umbo implant on a culture well insert was capable of producing cells in a continuous manner. (E, F) Showed rat TM epithelial cells with and without fibroblast feeder cells respectively. Scale bars = 500 mm. TMP, tympanic membrane perforation. DOI: 10.1089/scd.2018.0021 Different cells behave differently in culture Fibroblast-like cells tend to migrate individually at low densities because their main requirement is for substrate attachment and cell spreading. fibroblasts are the most motile cells at a low cell density (when cells are not in contact) Epithelial cells tend to grow in patches because they require cell–cell adhesion for optimum survival and growth. the whole patch may show signs of coordinated movement dense epithelial monolayers are the least motile Some cells can produce their own extracellular matrix (ECM) proteins to attach to the substrate e.g. fibrocytes secrete type I collagen and fibronectin into the matrix, whereas epithelial cells produce laminin. Some cells (such as stem cells) cannot synthesize their own ECM, so need coating on the culture surface the culture surface we provide must be coated with ECM proteins!