Introduction to animal cell culture

Introduction to animal cell culture

1. ANIMAL CELL CULTURE – INTRODUCTION DR. ANU P. ABHIMANNUE ASSISTANT PROFESSOR DEPARTMENT OF BIOTECHNOLOGY ST. MARY’S COLLEGE, THRISSUR 1 ANU P.A., ST. MARY'S COLLEGE, THRISSUR

2. INTRODUCTION Tissue culture is in vitro maintenance and propagation of isolated cells, tissues or organs in an appropriate artificial environment. The cells can be obtained directly from the host organism or may be derived from a cell line or cell strain that has already been established. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 2

3. DEVELOPMENT OF ANIMAL CELL CULTURE The first mammalian cell cultures date back to the early 20th century to study the development of cell cultures and normal physiological events such as nerve development. Ross Harrison in 1907 showed the first nerve fiber growth in vitro. In the 1950s, animal cell culture was performed at an industrial scale. Major development took place during polio epidemics during 1940s and 1950s and the accompanying requirement for viral vaccines. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 3 https://en.wikipedia.org/wiki/Ross_Granville_Harrison Ross Granville Harrison (1870 –1959), American biologist and anatomist

4. TYPES On the basis of the source of cells, animal cell culture is categorized into two types: Primary cell culture Secondary cell culture On the basis of the life span of culture, the cell culture are categorized into two types: Finite cell culture Continuous cell culture On the basis of the pattern of growth, the cell culture are categorized into two types: Monolayer culture Suspension culture ANU P.A., ST. MARY'S COLLEGE, THRISSUR 4

5. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 5 Primary cell culture Obtained straight from the cells of a host tissue. The cells dissociated from the parental tissue are grown on a suitable container and the culture thus obtained is called primary cell culture. Such culture comprises mostly hetergeneous cells Most of the cells divide only for a limited time. However , these cells are much similar to their parents. Secondary cell culture Sub-culturing of a primary culture by removing the growth media and disassociating the adhered cells produces secondary culture/cell-line/sub-clone. Leads to genotypic and phenotypic uniformity in the population. Most of the cells divide only for prolonged time. However, as they are sub-cultured serially, they become different from the original cell.

6. Advantages & Disadvantages - primary cell culture PROS: Primary cell culture represent the best experimental models for in vivo studies. Share the same karyotype as the parent. Express characteristics that are not seen in cultured cells. CONS: Difficult to obtain Limited lifespans . Potential contamination by viruses and bacteria ANU P.A., ST. MARY'S COLLEGE, THRISSUR 6

7. Advantages & Disadvantages - secondary cell culture PROS: Useful for obtaining a large population of similar cells Transformed to grow indefinitely. These cell cultures maintain their cellular characteristics. CONS: Cells have a tendency to differentiate over a period of time in culture and generate aberrant cells. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 7

8. SUSPENSION CULTURE Anchorage independent or non-adherent cells Do not attach to the surface of the culture vessel Grow floating in the culture medium giving rise to suspension culture Hematopoietic stem cells (derived from blood, spleen and bone marrow) and tumor cells. Grow much faster does not require frequent media replacement and can be easily maintained ANU P.A., ST. MARY'S COLLEGE, THRISSUR 8 http://www.techne.com/product.asp?dsl=206

9. MONOLAYER CULTURE Anchorage dependent/adherent cells Cells attach to solid/semi-solid substrate for proliferation Cover the bottom of the culture vessel with a continuous layer of cells, usually one cell in thickness, forming monolayer cultures Fibroblasts and epithelial cells are of such types Grow relatively slowly and require frequent replacement of the medium. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 9 https://www.semanticscholar.org/paper/Three-dimensional-cell-culture-systems-and-their-in-Edmondson Broglie/b776327dc8212170baef1e24100eaa4c60246ddb/figure/0

10. FINITE CELL LINES  The cell lines which go through a limited number of cell division having a limited life span are known as finite cell lines. These are slow growing cells (24–96 hours). Characterized by anchorage dependence and density limitation The cells passage several times and then lose their ability to proliferate and become senescence. Cell lines derived from primary cultures of normal cells are finite cell lines. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 10

11. CONTINUOUS CELL LINES  When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line. These are grown indefinitely as permanent cell lines and are immortal. Indefinite cell lines could be in vitro transformed cell lines or cancerous cells Can be grown in monolayer or suspension form Divide rapidly with a generation time of 12–14 hours ANU P.A., ST. MARY'S COLLEGE, THRISSUR 11

12. CONTINUOUS CELL LINES – PROPERTIES Less adherent Fast growing Less fastidious in their nutritional requirements Able to grow up to higher cell density and different in phenotypes from the original tissue. Potential to be subcultured indefinitely. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 12

13. CONTINUOUS CELL LINES ADVANTAGES Faster cell growth and achieve higher cell densities in culture. Serum-free and protein-free media are easily available. They have potential to be cultured in suspension in large-scale bioreactors. Easy to manipulate and maintain DISADVANTAGES Chromosomal instability Phenotypic variation in relation to the donor tissue, Change in specific and characteristic tissue markers ANU P.A., ST. MARY'S COLLEGE, THRISSUR 13

14. CONFLUENCY Confluence refers to the percentage of the surface of a culture dish that is covered by adherent cells. when cultured under appropriate conditions, cells occupy all of the available substrate i.e. reach confluence. For a few days, it can become too crowded for their container and this can be detrimental to their growth, generally leading to cell death if left for a long time. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 14 https://star-protocols.cell.com/protocols/107

15. PASSAGE NUMBER The passage number is the number of sub-cultures the cells have gone through. Passage number should be recorded and not get too high. High passage number is not advised because Passaging procedure is relatively stressful for adherent cells as they must be trypsinized . Hence, it is recommend not to passage cells more than once every 48 h. High passage number is avoided to prevent cells undergoing genetic drift and other variations. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 15

16. SPLIT RATIO Cell splitting is a means of increasing the capacity of a cellular system by subdividing or splitting cells into two or more smaller cells. For slow growing cells a low split ratio is recommended. Fast growing cells may require a high split ratio. Most cells must not be split more than 1:10 as the seeding density will be too low for the cells to survive. When the cells are approximately 80% confluent (80% of surface of flask covered by cell monolayer) they should still be in the log phase of growth and will require splitting/sub-culturing. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 16

17. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 17 https://twitter.com/kosheeka/status/1175391468430606336

18. GENERATION NUMBER It refers to the number of doublings that a cell population has undergone. It must be noted that the passage number and generation number are not the same, and they are totally different. Also known as, the population doubling (pd) number - indicates the number of cell generations the cell line has undergone i.e. the number of times the cell population has doubled. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 18

19. SUBCULTURE The transferring of portion of cells to a new vessel with fresh growth medium & more space and nutrients for the continual growth of both portions of cells. Sub-culturing keeps cells healthy and in a growing state. The primary culture, when sub-cultured, becomes a cell line or cell strain. Cell-line is grouped into two on the basis of the lifespan as finite or continuous. 19 ANU P.A., ST. MARY'S COLLEGE, THRISSUR

20. CELL - LINES Cell lines are used for Vaccine production Therapeutic proteins Pharmaceutical agents Anti-cancerous agents. For the production of cell lines, human, animal, or insect cells may be used. Chinese hamster ovary (CHO) is the most commonly used mammalian cell line. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 20

21. CELL - LINES When selecting a cell line, a number of general parameters must be considered, such as Growth characteristics Population doubling time Saturation density Plating efficiency Growth fraction Ability to grow in suspension.  ANU P.A., ST. MARY'S COLLEGE, THRISSUR 21

22. MORPHOLOGY OF CULTURED CELLS Different morphological structure of cells can be observed in a culture: Epithelium type Fibroblast type Lymphoblast type ANU P.A., ST. MARY'S COLLEGE, THRISSUR 22

23. EPITHELIUM TYPE Polygonal in shape Regular dimensions Becomes flattened as they attach to the substrate It grows as a continuous thin layer forming monolayer on solid surfaces ANU P.A., ST. MARY'S COLLEGE, THRISSUR 23 https://www.thermofisher.com/content/dam/LifeTech/migration/en/images/ics-organized/references/cell-culture-basics/data-image/180.par.20311.image.180.120.1.epithelial-like-gif.gif

24. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 24 https://cellapplications.com/fibroblast These are angular shaped elongated cells. It form an open network of cells rather than tightly packed cells These are either bipolar or multi-polar Anchorage-dependent cells and often align into parallel assemblies FIBROBLAST TYPE

25. LYMPHOBLAST TYPE Cultured cells show a spherical outline They are typically grown in suspension ANU P.A., ST. MARY'S COLLEGE, THRISSUR 25

26. REQUIREMENTS – PLASTICWARE AND GLASSWARES Most of the consumables are commercially available as single use, sterile packs. The use of plastic-ware is preferred over recycling glassware because Cost effective Enables a higher level of quality assurance Removes the need for validation of cleaning and sterilization procedures. Designed according to the needs. Pre - treated to provide a hydrophilic surface for anchorage dependent cells or untreated for suspension culture. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 26

27. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 27

28. SPINNER FLASK Flask for animal cell culturing and it provides better cell production and viability than conventional flasks Has a top Cap and 2 Angled Sidearms Side baffles enhance aeration and agitation of flask contents impeller design ensures optimal stirring ANU P.A., ST. MARY'S COLLEGE, THRISSUR 28 https://www.sigmaaldrich.com/catalog/product/sigma/cls450236l?lang=en&region=IN

29. ROLLER BOTTLES Cylindrical vessels that revolve slowly (5-60 revolutions per hour) enabling culturing of adherent cells in large quantity. Roller bottles are economical and provides larger surface areas for the growth. The gentle agitation prevents formation of gradients in the medium It also helps in superior gas exchange as only a thin layer of medium covers the cell. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 29 https://www.bioprocessonline.com/doc/cell-culture-roller-flask-system-cellroll-0001

30. T-FLASK Animal cell culture flask with an upright lid for prevention of contamination. Usually has vented caps (with breathable membrane) or even closed caps are available Non- Pyrogenic , DNase / RNase -FREE It is available in various capacities like T25, T75, T175 and T225. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 30 https://www.tpp.ch/page/produkte/02_a_zellkultur_flasche_wv.php

31. CELL CULTURE DISHES Cell culture dishes are disposable/reusable shallow containers Dishes can be pre-treated/ untreated for the growth of anchor-dependent/ suspension cells. They come in a variety of sizes in single- or multi-well formats, and they can be round or rectangular. made of borosilicate glass/polystyrene or polycarbonate plastics and allows distortion-free microscopic observation. Lids provide consistent gas exchange while offering protection from the environment. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 31 https://www.amazon.com/Culture-60x15mm-Treated-Sterile-Sleeve/dp/B075JQL9HZ

32. Apart from the specific glasswares , animal cell culture make use of the common apparatus like Measuring jar Tubes/vials Tissue grinder/homogenizer Media bottle Flasks etc.. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 32

33. ADVANTAGES Physiochemical and physiological condition: Role and effect of pH, temperature, O 2 /CO 2  concentration, and osmotic pressure of the culture media can be altered to study their effects on the cell culture Metabolism of cell: To study cell metabolism, investigate the physiology and biochemistry of cells. Cytotoxic assay: Effect of various compounds or drugs on specific cell types such as liver cells can be studied. Homogenous cultures: These cultures help study the biology and origin of the cells ANU P.A., ST. MARY'S COLLEGE, THRISSUR 33

34. ADVANTAGES Valuable biological data from large-scale cell cultures: Specific proteins can be synthesized in large quantities from genetically modified cells in large-scale cultures. Consistency of results: Reproducibility of the results that can be obtained by the use of a single type/ clonal population. Identification of cell type: Specific cell types can be detected by the presence of markers such as molecules or by karyotyping . Ethics: Ethical, moral, and legal questions for utilizing animals in experiments can be avoided. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 34

35. DISADVANTAGES Expenditure and expertise: This is a specialized technique that requires aseptic conditions, trained personnel, and costly equipment. Dedifferentiation: Cell characteristics can change after a period of continuous growth of cells in cultures, leading to differentiated properties compared to the original strain. Low amount of product: The miniscule amount of mAB and recombinant protein produced followed by downstream processing for extracting pure products increases expenses tremendously. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 35

36. DISADVANTAGES Contamination: Mycoplasma and viral infection are difficult to detect and are highly contagious. Instability: Aneuploidy chromosomal constitution in continuous cell lines leads to instability. In addition, this system cannot replace the complex live animal for testing the response of chemicals or the impact of vaccines or toxins. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 36

37. APPLICATIONS Vaccines Production cells are widely cultured on a large scale to produce vaccines for many diseases like rabies, chickenpox, hepatitis B, polio and measles. Virus cultivation and study It is easy to observe cytopathic effects and easy to select particular cells on which the virus grow as well as to study the infectious cycle. Cell lines are convenient for virus research because cell material is continuously available. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 37

38. Cellular and molecular biology providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of different toxic compounds on the cells, and mutagenesis and carcinogenesis. In   Cancer   Research Normal cells can be transformed into cancer cells by methods including radiation, chemicals, and viruses. These cells can then be used to study cancer more closely and to test potential new treatments. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 38

39. Gene therapy Cells having a functional gene can be replaced to cells which are having non-functional gene, and for which the cell culture technique is used. Immunological studies Cell culture techniques are used to know the working of various immune cells, cytokines, lymphoid cells, and interaction between disease-causing agents and the host cells. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 39

40. Cell lines are also used in In-vitro fertilization (IVF) technology Recombinant protein development Drug selection and improvement. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 40

41. NAMING A CELL LINE Proper Nomenclature of Cell Lines is vital Because number of cell lines has dramatically increased To maintain individuality of cell lines Avoid confusion and duplication of cell line names and identities. For consistent usage in publications. Cell lines are designated with an alphanumeric code for their identification. For instance, the code NHB 2-1 represents the cell line from normal human brain, followed by cell strain (or cell line number) 2 and clone number 1. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 41

42. The usual practice in a culture laboratory is to maintain a log book or computer database file for each of the cell lines. While naming the cell lines, it is absolutely necessary to ensure that each cell line designation is unique so that there occurs no confusion when reports are given in literature. At the time of publication, the cell line should be prefixed with a code designating the laboratory from which it was obtained NCI for National Cancer Institute Wl for Wistar Institute MCF for Michigan Cancer Foundation ANU P.A., ST. MARY'S COLLEGE, THRISSUR 42

43. REFERENCES https://www.abcam.com/ps/pdf/protocols/cell_culture.pdf https://www.corning.com/catalog/cls/documents/application-notes/CLS-AN-042.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325846/ https://microbeonline.com/animal-cell-culture-introduction-types-methods-applications/ https://academicjournals.org/journal/BMBR/article-full-text-pdf/7A2154261212 https://www.qiagen.com/us/service-and-support/learning-hub/molecular-biology-methods/animal-cell-culture/ https://microbeonline.com/animal-cell-culture-introduction-types-methods-applications/ https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/maintaining-cultured-cells.html#:~:text=Subculturing%2C%20also%20referred%20to%20as,cell%20line%20or%20cell%20strain. ANU P.A., ST. MARY'S COLLEGE, THRISSUR 43