ANIMAL BIOTECHNOLOGY. Cell culture and its types

Biotechnology

1. ANIMAL BIOTECHNOLOGY

2. Cell culture - culture derived from dispersed cells taken from original tissue/ primary culture/ cell line /cell strain by enzymatic, mechanical, or chemical disaggregation. Primary cell culture Obtained directly from the cells of the host tissue. The cells ---dissociated from the parental tissue --- grown and the culture thus obtained is called primary cell culture. Comprises mostly heterogeneous cells Most of the cells divide only for a limited time; much similar to their parental tissues. May grow either as an adherent monolayer or in a suspension . Adherent cells Anchorage-dependent, propagate as a monolayer. Need to be attached to a solid or semi-solid substrate for proliferation. Adherence -extracellular matrix (from tissues of organs that are immobile and embedded in a network of connective tissue). Exception- hematopoietic cells. Examples: Fibroblasts and epithelial cells.

3. When the bottom of the culture vessel is covered with a continuous layer of cells (i.e. one cell in thickness) - monolayer cultures. Ex: continuous cell lines . Can be transferred directly and observed under microscope. Suspension cells Do not attach; cells can survive and proliferate without attachment Also called anchorage- independent or non-adherent cells Can be grown floating in the culture medium. Ex: Hematopoietic stem cells (derived from blood, spleen, and bone marrow) and tumor cells. Grow much faster Do not require the frequent replacement of the medium Can be easily maintained Homogeneous types and enzyme treatment is not required for the dissociation of cells Have a short lag period.

4. After the cells are isolated and proliferated on a suitable media----use up all the available substrates---- reach CONFLUENCE There arise the need for SUBCULTURING i.e. a portion of cells is transferred to a new vessel with a fresh growth medium Provides more space and nutrients for the continual growth of cells. Keeps cells healthy and in a growing state. A passage number  refers specifically to how many times a cell line has been sub-cultured. Gives a general indication of how old the cells may be for various assays. CLASSIFICATION BASED ON LIFE SPAN OF CULTURE CELL LINES These are the cultures of animal cells that can be propagated repeatedly and sometimes indefinitely when given appropriate fresh medium and space. Also refers to the propagation of culture after the first subculture. Usually become immortalized Contains several cell lineages of either similar or distinct phenotypes.

5. FINITE CELL LINES Cell lines go through a limited number of cell divisions Normally divide 20 to 100 times, doubling time- 24-96h Have limited life span, grow slowly and form monolayer The cells passage several times and then lose their ability to proliferate and reaches senescence (a genetically determined event) Cell lines derived from primary cultures of normal cells are finite cell lines Display anchorage independence, contact inhibition and density limitation secondary culture - Obtained by subculturing of primary culture Secondary cell culture and cell line Also called as secondary cell line , or sub-clone. I nvolves removing the growth media and disassociating the adhered cells (usually enzymatically). L eads to the generation of cell lines. During the passage, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. When sub-cultured serially, they become different from the original cell.

6. CONTINUOUS CELL LINES  When a finite cell line undergoes transformation and acquires the ability to divide indefinitely and rapidly (12-24h), it becomes a continuous cell line Usually aneuploid and often have a chromosome number between the diploid and tetraploid values C an occur spontaneously or can be chemically or virally induced or from the establishment of cell cultures from malignant tissue. C an be sub-cultured and grown indefinitely as permanent cell lines and are immortal . A re 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. Usually grow more in suspension. H ave a tendency to grow on top of each other in multilayers on culture-vessel surfaces.

7. Nomenclature of Cell Lines Must be unique NHB 2-1 represents the cell line from normal human brain, followed by cell strain (or cell line number) 2 and clone number 1. LT156 -lung tumor biopsy number 156. For finite cell lines, the number of population doublings should be estimated and indicated after a forward slash, e.g., NHB2/2, and increases by one for a split ratio of 1:2 (e.g., NHB2/2, NHB2/3, etc.), by two for a split ratio of 1:4 (e.g., NHB2/2, NHB2/4, etc.), and so on. When dealing with a continuous cell line a ‘‘p’’ number at the end is often used to indicate the number the number of passages since the last thaw from the freezer e.g., HeLa-S3/p4. Essential to maintain a log book or computer database file for each of the cell lines.

8. While naming the cell lines, it is absolutely necessary to ensure that each cell line designation is free of confusion. Should be numbered if there are more than one cell line Further, at the time of publication, the-cell line should be prefixed with a code designating the laboratory from which it was obtained e.g. NCI for National Cancer Institute, Wl for Wistar Institute. EXAMPLES OF COMMON CELL LINES MCF-7 (breast cancer), HL 60 (leukemia), HeLa (human cervical cancer cells), WI-38 human embryonic lung fibroblasts, BHK21 baby hamster kidney fibroblasts,

9. PROPERTIES OF FINITE AND CONTINUOUS CELL LINES

10. Cell strain These are the lineage of cells originating from the primary culture (OR)w hen cells are selected from a culture, by cloning or by any other method, the resulting subline is called as cell strain E ither derived from a primary culture or a cell line by the positive selection / cloning of cells with specific properties or characteristics. O ften acquires additional genetic changes subsequent to the initiation of the parent line.

11. HISTORY AND DEVELOPMENTS OF ANIMAL CELL CULTURE 1907: Frog embryo nerve fiber outgrowth in vitro 1912 : Explants of chick connective tissue; heart muscle contractile for 2–3 months 1916 :Trypsinization and subculture of explants 1925–1926: Differentiation in vitro in organ culture 1940s: Introduction of use of antibiotics in tissue culture 1948: Cloning of the L-cell 1949: Growth of virus in cell culture 1952–1955 : Establishment the first human cell line, HeLa, from a cervical carcinoma 1954: Fibroblast contact inhibition of cell motility, Salk polio vaccine grown in monkey kidney cells Late 1950s: Realization of importance of mycoplasma (PPLO) infection

12. 1961 : Definition of finite life span of normal human cells , Cell fusion–somatic cell hybridization 1964–1969: Rabies, Rubella vaccines in WI-38 human lung fibroblasts 1967: Epidermal growth factor 1968: Retention of differentiation in cultured normal myoblasts, Anchorage-independent cell proliferation 1973: DNA transfer, calcium phosphate 1980s: Regulation of gene expression, Oncogenes, malignancy, and transformation 1983: Reconstituted skin cultures 1990s : Industrial-scale culture of transfected cells for production of biopharmaceuticals 1998 : Tissue-engineered cartilage 1998 : Culture of human embryonic stem cells 2000+ : Human Genome Project: genomics, proteomics, genetic deficiencies and expression errors, tissue engineering

13. REQUIREMENTS OF ANIMAL CELL CULTURE LABORATORY Important to maintain asepsis Must be dust free and have no through traffic. Important considerations: Ventilation, accommodation, Renovations, access and quarantine Small scale Medium scale Large scale

14. DESIGN FOR A SMALL ANIMAL TISSUE CULTURE LABORATORY

15. MEDIUM SIZED TISSUE CULTURE LABORATORY

16. TISSUE CULTURE FACILITIES

17. REQUIREMENTS OF A ANIMAL TISSUE CULTURE LABORATORY

18. Inverted microscope Automatic dispenser Plate filler Multipoint pipettor Plate reader

19. CO2 incubator OTHER REQUIREMENTS: Bench top autoclave Free standing autoclave Sterilizing and drying ovens Millipore water filtration unit Glass ware washing machine Weighing balance pH meter Refrigerators and freezers for storage Cryostorage containers Culture vessels Disinfectants

20. LAMINAR AIR FLOW CABINET MULTIWELL PLATES

21. TYPES OF TISSUE CULTURE

22. PROPERTIES OF DIFFERENT TYPES OF CULTURE

23. ADVANTAGES OF TISSUE CULTURE Control of the Environment Characterization and Homogeneity of Sample Economy, Scale, and Mechanization In Vitro Modeling of In Vivo Conditions LIMITATIONS Lack of expertise Origin of cells Instability Dedifferentiation and selection

24. PHYSICAL CONDITIONS FOR CULTURING ANIMAL CELLS DEVELOPMENT OF MEDIA Previously used media – derived based on tissue extracts Widely adopted media - Eagle’s Basal Medium and Eagle’s Minimal Essential Medium (MEM) Media- has to be optimized and modified for specific conditions For cells of specific lineage – selective serum free media For cells grown for the formation of products , as hosts for viral propagation, or for non-cell-specific molecular studies rely mainly on Eagle’s MEM Media- be able to reduce the risk of contamination

25. PHYSICOCHEMICAL PROPERTIES OF MEDIA pH – important for studying growth, plating efficiency or special function analysis For most of the cell lines- pH is 7.4 For some normal fibroblast -pH 7.4–7.7 For transformed cells -pH 7.0–7.4 For epidermal cells - pH 5.5 (not universally accepted) Commonly used indicator- Phenol red Red at pH 7.4, becomes orange at pH 7.0, yellow at pH 6.5, lemon yellow below pH 6.5, more pink at pH 7.6, and purple at pH 7.8 CO 2 and Bicarbonate – Carbon dioxide in the gas phase dissolves in the medium, establishes equilibrium with HCO3 − ions, and lowers the pH. CO 2 can be replaced by buffers (HEPES pH range 7.2-7.6) The inclusion of pyruvate -increase in endogenous production of CO 2 No exogenous CO 2 , HCO 3 − is required Ex: Leibovitz L15 medium contains a higher concentration of sodium pyruvate (550 mg/L) but lacks NaHCO 3 and does not require CO 2 in the gas phase

26. Cultures in open vessels need to be incubated in an atmosphere of CO 2 The concentration is in equilibrium with the sodium bicarbonate in the medium Cells at moderately high concentrations (≥1 × 10 5 cells/mL) and grown in sealed flasks –No CO 2 added to the gas phase-bicarbonate concentration is kept low (∼4 mM) True if the cells are high acid producers. At low cell concentrations (cloning and with some primary cultures) it is necessary to add CO 2 to the gas phase of sealed flasks. When venting is required, to allow either the equilibration of CO 2 or its escape in high acid producers, it is necessary to leave the cap slack or to use a CO 2 -permeable cap. Buffering – to stabilize pH Culture media must be buffered under two sets of conditions: (1) open dishes, wherein the evolution of CO 2 causes the pH to rise (2) overproduction of CO 2 and lactic acid in transformed cell lines at high cell concentrations, when the pH will fall. Sometimes, exogenous CO 2 may be required by some cell lines, particularly at low cell concentrations, to prevent the total loss of dissolved CO 2 and bicarbonate from the medium.

27. Oxygen Under in vivo conditions- required for cellular respiration For cultured cells - dissolved O 2 , which can be toxic due to the elevation in the level of free radicals Correct O 2 –required for respiration + overcoming toxicity Use of glutathione, 2-mercaptoethanol ( β- mercaptoethanol ) or dithiothreitol into the medium Most cell culture – lower oxygen tension For late-stage embryos, new - borns , or adults, require up to 95% O 2 in the gas phase Most dispersed cell cultures prefer lower oxygen tensions Requirement for selenium in medium - related to oxygen toxicity Selenium –cofactor in glutathione synthesis Oxygen tolerance + selenium may be provided by serum important factor in determining O2 tension

28. Osmolality Usually measured by depression of the freezing point or elevation of the vapor pressure, of the medium Most cultured cells exhibit tolerance for osmotic pressure Osmolality of human plasma is about 290 mosmol /kg Osmolalities between 260 mosmol /kg and 320 mosmol /kg- acceptable for most cells Must be kept consistent at ±10 mosmol /kg Slightly hypotonic medium may be better for Petri dish or open-plate culture to compensate for evaporation during incubation Addition of HEPES and drugs dissolved in strong acids and bases and their subsequent neutralization -affect osmolality

29. Temperature The optimal temperature for cell culture is dependent on The body temperature of the animal from which the cells were obtained Any anatomic variation in temperature The incorporation of a safety factor to allow for minor errors in regulating the incubator Optimum temperature is 37 C For birds, avian cells should be maintained at 38.5 C Cultured mammalian can survive several days at 4 C, can be frozen and cooled to −196 C. Small change in temperature- change in pH pH has to be adjusted to 0.2units lower at room temperature

30. Viscosity Important during trypsinization and cell suspension influenced mainly by the serum content Have little effect on cell growth Adjusting viscosity – helps in reducing cell damage Addition of carboxymethylcellulose (CMC) or polyvinylpyrrolidone (PVP) useful in increasing viscosity Surface Tension and Foaming Foaming- increases the rate of protein denaturation Also limit gaseous diffusion Caused when 5% CO 2 in air is bubbled through medium containing serum in stirrer vessels/bioreactors Addition of a silicone antifoam/ Pluronic F68 at the concentration of 0.01–0.1%, helps to prevent foaming and protect cells against shear stress from bubbles

31. BALANCED SALT SOLUTION (BSS) Composed of inorganic salts and may include sodium bicarbonate and, in some cases glucose HEPES buffer (5–20 mM) may be added to these solutions if necessary and the equivalent amount of NaCl omitted to maintain the correct osmolality Also used as a diluent for concentrates of amino acids and vitamins to make complete media As an isotonic wash or dissection medium , and for short incubations up to about 4 h (in presence of glucose) The choice of BSS is dependent on both the CO 2 tension and the intended use of the solution for tissue disaggregation or monolayer dispersal Also dependent on whether the solution will be used for suspension culture of adherent cells BSS recipes are often modified- omitting glucose/phenol red from Hank’s BSS OR leaving Ca2+ and Mg2+ from Dulbecco’s PBS (i.e. PBS solution A) HBSS, EBSS, and PBS rely on the relatively weak buffering of phosphate HEPES (10–20 mM) - most effective buffer in the pH 7.2–7.8 range, and Tricine in the pH 7.4–8.0 range

32. COMPLETE MEDIA A media that has all its constituents and supplements added and is sufficient for the use specified Some components (ex: serum, growth factors, glutamine) has to added just before use The complex media contain different amino acids, including nonessential amino acids and additional vitamins Media often supplemented with extra metabolites (e.g., nucleosides, tricarboxylic acid cycle intermediates, and lipids) and minerals. Defined simple media- ex: Eagle’s MEM Defined complex media- ex: Medium 199 (M199), CMRL 1066, RPMI 1640, F12 media Nutrient concentrations – low in F12 and high Eagle’s MEM

33. Amino acids The essential amino acids + cystine and/or cysteine, arginine, glutamine, and tyrosine are required based on the type of cell being cultured Sometimes non-essential amino acids are to be added Concentration of amino acids- limits the maximum cell concentration attainable in turn affects cell survival and growth rate. Glutamine – has to be added Serves as source of energy and carbon Glutamax is a alanyl-glutamine dipeptide which is more stable than glutamine Vitamins – dependent on cell line Eagle’s MEM – water-soluble vitamins (the B group, choline, folic acid, inositol, and nicotinamide – No biotin Other requirements of media- compensated by serum All the fat-soluble vitamins (A, D, E, and K) only in M199, whereas vitamin A is present in LHC-9 and vitamin E in MCDB 110 Fischer’s medium has a high folate concentration Concentration of choline and nicotinamide – increased in serum free media

34. Salts Majorly Na + , K +, Mg 2+ , Ca 2+ , Cl−, SO 4 2− , PO 4 3− , and HCO 3 − Contribute to osmolality Most media derive their salt concentration from Earle’s BSS (high bicarbonate, % CO2) or Hank’s BSS (low bicarbonate, gas phase- air) Calcium ions- for cell adhesion and signal transduction, minimizes cell aggregation and attachment Affects cell proliferation /differentiation Na + , K + , Cl - - regulate membrane potential Glucose Serves as a source of energy Organic supplements Proteins, peptides, nucleosides, citric acid cycle intermediates, pyruvate, and lipids – helps in cloning and maintenance of cell lines Hormones and growth factors antibiotics

35. Initially introduced to reduce the frequency of contamination Disadvantages encourage antibiotic resistance Hide the presence of low-level, cryptic contaminants that can become fully operative if the antibiotics are removed or the culture conditions change or resistant strains develop They may hide mycoplasma infections They have antimetabolic effects that can cross-react with mammalian cells They encourage poor aseptic technique Recommended – not to use antibiotics or to be used during primary culture or large-scale labor-intensive experiments with a high cost of consumables Antibiotics used in animal cell culture- effective in eliminating bacterial contaminations

36. Serum contains growth factors, which promote cell proliferation, and adhesion factors and antitrypsin activity Source of minerals, lipids, hormones Commonly used sera- bovine calf, fetal bovine, adult horse, and human serum Calf (CS) and fetal bovine (FBS) – most widely used Human serum- screened for virus before usage Horse serum - less likely to metabolize polyamines, due to lower levels of polyamine oxidase (polyamines are mitogenic for some cells) More consistent Protein Major components of serum, required for cell attachment and growth Carriers for minerals, fatty acids, and hormones Albumin- lipids and minerals, globulin; fibronectins- promotes cell attachment Fetuin - enhances cell attachment; transferrin- binds iron, making it less toxic and bioavailable Proteins - increases the viscosity of the medium, reducing shear stress during pipetting and stirring, and may add to the medium’s buffering capacity.

37. Growth factors Present in small amounts PDGF stimulates growth in fibroblasts and glia, but other platelet-derived factors, such as TGF-β, may inhibit growth or promote differentiation in epithelial cells Fibroblast growth factors (FGFs), Epidermal Growth factor (EGF), Vascular endothelial growth factor (VEGF), angiogenin, insulin-like growth factors IGF-I and IGF-II Hormones Insulin promotes the uptake of glucose and amino acids Fetal serum + somatomedins – have a mitogenic effect Hydrocortisone – promote cell attachment and cell proliferation It may be sometimes cytoststic - inducing differentiation Nutrients and Metabolites Lipids - Helps in binding proteins Ex: Linoleic acid, oleic acid, ethanolamine, and phosphoethanolamine Minerals- Fe, Cu, Zn, Se- detoxifies free radicals Inhibitors – Heat inactivation, TGF- β

38. Other supplements Amino Acid Hydrolysates Bactopeptone , tryptose , and lactalbumin hydrolysate – proteolytic digests of beef heart or lactalbumin Mainly contain amino acids and small peptides Bactopeptone , tryptose – also contain nucleosides and other heat-stable tissue constituents (fatty acids and carbohydrates) Embryo extract Was originally used as a component of plasma clots Crude homogenate of 10-day-old chick embryo – centrifuged Contained both low and high molecular weight fraction low-molecular-weight fraction promoted cell proliferation The high-molecular-weight fraction promoted pigment and cartilage cell differentiation Contain growth factors, proteoglycans, peptides and other constituents Conditioned medium Necessary for growth and differentiation Uses feeder layer- release of collagen Also contain fibronectin, and proteoglycans and growth factors, heparin-binding group (FGF, etc.), insulin-like growth factors (IGF-I and -II), PDGF, and several others

39. Serum-free Media Disadvantages of serum in the media - Physiological variability, shelf life and consistency, quality and control, specificity, availability, downstream processing, contamination, cost effectiveness, growth inhibitors and standardization Hence, there existed a need for the development of serum-free media ADVANTAGES Selective media - ability to make a medium selective for a particular cell type Regulation of Proliferation and Differentiation – ability to switch from growth enhancing medium to differentiation-inducing medium DISADVANTAGES Multiplicity of media Selectivity Reagent purity Cell proliferation Availability

40. Serum can be replaced in a culture by Adhesion factors- fibronectins and laminins /pre-treating plastics with poly-L-lysine (1 mg/mL) enhanced cell survival Protease inhibitors- soya bean trypsin inhibitor or 0.1 mg/mL aprotinin Purified porcine trypsin can be used Hormones – as a replacement for serum Somatotropin at 50 ng/mL, insulin at 1–10 U/mL, hydrocortisone – enhances cloning efficiency in glial cells and fibroblasts 10 pM triiodothyronine (T3) for the culture of lung epithelial cells Various combinations of estrogen, androgen, or progesterone with hydrocortisone and prolactin at a concentration of 10 nM can be shown to be necessary for the maintenance of mammary epithelium Growth factors- EGF, PDGF, interleukins @ 1–10 ng/mL range Keratinocyte growth factor induce proliferation and differentiation in prostatic epithelium Hepatocyte growth factor (HGF) - mitogenic for hepatocytes + morphogenic for kidney tubules

41. Have localized and limited range of action Nutrients - Iron, copper, and a number of minerals have been included in serum-free recipes Selenium (Na 2 SeO 3 ), at 20 nM concentration acts as precursor for lipids Proteins and polyamines - tissue extracts and BSA @ 1–10 mg/mL Transferrin @ 10 ng/mL-carrier for iron acts as mitogen Putrescine @ 100 nM concentration Matrix – fibronectin / polylysine SELECTION OF SERUM-FREE MEDIA – according to originator’s recommendation Commercially available media- MCDB 131 for endothelial cells; LHC-9 for bronchial epithelium; Opti-MEM for hematopoietic cells

42. DEVELOPMENT OF SERUM FREE MEDIUM To take a known recipe for a related cell type, with or without 10–20% dialyzed serum Alter the constituents individually or in groups Reduce the serum, until the medium is optimized according to particular requirement If a group of compounds is found to be effective in reducing serum supplementation, the active constituents may be identified by the systematic omission of single components and then the concentrations of the essential components optimized – TIME CONSUMING PROCESS Supplementing existing media such as RPMI 1640 or combining media such as Ham’s F12 with DMEM Restricting the manipulation of the constituents to a shorter list of substances- minerals and growth factors PREPARATION OF SERUM FREE MEDIUM – similar to regular media preparation Using Ultrapure reagents and water; take care of Fe 2+ and Ca 2+ Cations in stock solutions should be kept at a low pH and added at last; phosphate free Autoclave/ filter sterilization; better to prepare stock solutions Growth factors, hormones, and cell adhesion factors are added separately just before the medium is used

43. PREPARATION AND STERILIZTION

44. GLASSWARES- Do not let soiled glassware dry out Sodium hypochlorite, should be included in the water used to collect soiled glassware a) to remove any potential biohazard and b) to prevent microbial growth in the water Selection of effective detergent Rinse thoroughly before drying the glasswares Use inverted position for drying glasswares Sterilize the glassware by dry heat to minimize the risk of depositing toxic residues from steam sterilization Bottles may be loosely capped with screw caps and foil, tagged with autoclave tape, and autoclaved All new apparatus and materials (silicone tubing, filter holders, instruments, etc.) should be soaked in detergent overnight, thoroughly rinsed, and dried. Corrosive materials- must be washed directly Used items- rinsing and immersing in detergent Proper wrapping- important Plastics- immersed in 70% alcohol, followed by UV treatment Sometimes- ethylene oxide treatment; gamma irradiation @ 25 kGy .

45. STERILIZING REAGENTS AND MEDIA All constituents must be properly dissolved Concentrated media are often prepared at a low pH (between 3.5 and 5.0) Commercial media may be available as working-strength solutions (1×) with or without glutamine 10× concentrates, usually without NaHCO 3 and glutamine – expensive but convenient Powdered media, with or without NaHCO 3 and glutamine – has growth promoting activities; poor sterility Equilibrate and check pH @ 37 O C Adjusting pH- During the first time, add the stipulated amount of NaHCO 3 , allow samples with varying amounts of alkali to equilibrate overnight at 37 O C in the appropriate gas phase Check pH in the following morning NaHCO 3 - helpful in establishing equilibrium with atmospheric CO 2

46. Eagle’s MEM with Earle’s salts- require high NaHCO 3 Changing concentration- make sure to maintain perfect osmolality Additions to media – HEPES, glutamine (stored in frozen condition) Media- checked for quality control BSS- sterilized by autoclaving- glucose and bicarbonate has to be omitted Customized medium- media made up in house from individual constituents Better to make concentrated stocks always Using sterilizing filter of 0.2-µm porosity, and diluted with high- or low-bicarbonate BSS Advantages- extra nutrients (oxo-acids, nucleosides, minerals, etc ) can be added

47. STERILIZATION OF MEDIA Autoclavable media much less labor intensive, less expensive and lower failure rate than filtration The medium is buffered to pH 4.25 with succinate, in order to stabilize the B- vitamins during autoclaving, and is subsequently neutralized Preferred - adding sterile glutamax after autoclaving BSS is autoclavable without glucose and glutamine Various amino acid hydrolysates ( tryptose phosphate broth and other microbiological media) are sterilizable by autoclaving Sterile filtration – use of 0.1- 0.2-µm microporous filters for heat labile constituents Materials – polyethersulfone (PES), nylon, polycarbonate, cellulose acetate, cellulose nitrate, polytetrafluorethylene (PTFE) and ceramics Size ranging from syringe fitting filters- small and intermediate in-line filters – multi disk and cartridge filters PES filters- fast flowing; Polycarbonate filters- have uniform porosity, helpful in maintaining uniform flow rate

48. FILTRATION Positive- from a pressurized container Using peristaltic pump Negative pressure filtration- simpler Useful for small scale operations 50-500mL can be filtered Filter flasks- 150-1000mL capacity DISPOSABLE FILTERS simple disk filters hollow-fiber units Cartridges REUSABLE FILTERS Uses membranes/cartridges Can be sterilized by autoclaving Positive pressure- recommended

49. PREPARATION OF SERUM Difficulty arises during filtration Small-scale serum processing: After clot retraction Small volumes of serum may be centrifuged (5–10,000 g) and then filtered through a series of disposable filters (e.g., 50-mm Millipore Millex or Pall Gelman Acrodisc ) Final filtration through a 50-mm, 0.1-µm porosity sterile disposable filter Graded series of filters can also be used after centrifugation Storage- in bottles, to be used after 2-3 weeks post thawing Best used within 6–12 months of preparation (-20 O C storage) Use of Polycarbonate or high-density polypropylene bottles if samples are stored at -70 O C Prepared media is checked for quality control and sterility testing Sterility testing – bubble point, incubation, downstream secondary filtration Storage of media - media made up without glutamine should last 6–9 months at 4◦C If added – 2-3 weeks Incandescent lighting should be used in cold rooms Bottles of medium should not be exposed to fluorescent lighting Dark freezer for long- term storage

50. SCALE-UP OF CULTURE Stirrer culture / spinner culture - simple culture systems at the low end of the laboratory range of equipment Fermentor - for bacteria and yeast , 50-100L capacity Scale- up depends on cell proliferation in suspension or anchored to substrate Monolayer- more complex SCALE-UP IN SUSPENSION Mainly involves increase in the volume of culture medium Agitation required (when depth >5mm) + sparging with CO 2 , air + adequate gas exchange For smaller surface- stirring with the help of magnet; for larger ones- using large surface area paddle Stirring – 30-100rpm Antifoam agents 0.01-0.1% concentration Necessary to maintain viscosity (use of CMC)

51. Media- must be present in bags- must allow proper gas exchange hooked up alongside the stirrer flask, warmed to 37 O C and allowed to run in unattended cases Make up the media concentrates Harvesting – involves a bottom outlet Problem- clogging Best method- pumping/displacement Remove the in-line filter from the gas line and add silicone tubing and either siphon off the cells or use a peristaltic pump, tilting the vessel when the fluid is near to the bottom Disconnect the 5% CO 2 supply, replace the micropore filter with a flexible tube, attach the 5% CO 2 supply to the other port, and blow the cells out through the gas line. CONTINUOUS CULTURE If the cells are to be maintained at set of concentrations- use of biostat /chemostat Cells grown in mid-log phase- cells harvested- replaced with fresh medium (or) Cells run off continuously at constant rate- addition of media at the same rate- regulation of flow rate by peristaltic pump

52. For bulk production (1-20L )- batch method But necessary to maintain steady state For 50-1000L range, continuous culture is best For adherent cells- cannot be present in suspension Exceptional cases- use of microcarriers, transformed cells Culture vessels- coated with water repellent (e.g., Repelcote ) Calcium concentration may need to be reduced S-MEM medium -a variation of Eagle’s MEM with no calcium in the formulation and has been used for the culture of HeLa-S3 and other cells in suspension SCALE AND COMPLEXITY For 10L culture- Standard bench-top stirrer cultures – use of controlled fermentor /bioreactor MIXING AND AERATION Use of slowly rotating large-bladed paddle with a relatively high surface area

53. Use of air lift fermentors – upto 20,000L capacity BelloCell aerator culture – minimum shear

54. Perfused suspension culture – based on compartmentalization Also uses a hollow fibres with double concentric system- nutrients collected at the centre, products on the outer space and cells in between two Use of fluidised bed reactors

55. SCALE UP IN MONOLAYER Large surface area is required Nunc cell factory: has rectangular Petri dish-like units, S.A- 600–24,000 cm 2 , interconnected at two adjacent corners by vertical tubes Initially designed for harvesting supernatant

56. Multiarray Disks, Spirals, and Tubes Mainly to increase the surface area for a monolayer culture Use of multisurface perfusion system ( CellCube ) Consists of a hollow polystyrene cube with multiple inner lamellae, perfused with oxygenated, heated medium The inner lamellae are capable of supporting monolayer growth on both surfaces Roller cultures Cells in a round bottle or tube is rolled If the cells are adhesive, they will gradually attach to the inner surface of the bottle and grow to form a monolayer ADVANTAGES The increase in utilizable surface area for a given size of bottle Constant, but gentle, agitation of the medium The increased ratio of the medium’s surface area to its volume

57. Microcarriers Use of microbeads; 90-300μm in diameter Made of plastic, glass, gelatin or collagen gives a maximum ratio of the surface area of the culture to volume of the medium Stirring without grinding beads is essential Agitation with the help of suspended rotating pendulum/paddle Macrocarriers Porous carriers that are larger with a macroscopic structure Made up of polylactic acid (PLA), polyglycolic acid (PGA), collagen, or gelatin ( Gelfoam ) in a variety of different geometries These can be loaded with cells and stirred in a bioreactor or perfused in a fixed-bed or fluidized bed reactor Fibracel is one such product designed for use in the Celligen bioreactor or a stirred bioreactor.

58. Perfused monolayer culture To facilitate medium replacement and product recovery Ex: CellCube Membrane perfusion The culture bed is a flat, permeable sheet Ex: Membroferm Hollow fiber perfusion Adherent cells grow on the outer surface of the perfused microcapillary bundles High molecular-weight products concentrate in the outer space with the cells, while nutrients are supplied and metabolites removed via the inner space Re-creation of high, tissue like cell densities, matrix interactions, and the establishment of cell polarity Can also be used for hybridomas Fixed bed and fluidised bed reactors Microencapsulation – use of sodium alginate – nutrients, metabolites, and gas freely permeate the gel, macromolecules trapped inside Product recovery- reducing the concentration of divalent cations