1. Introduction to Mammalian Cell Culture
Lecture 1
Introduction to Mammalian Cell Culture

2. LECTURE CONTENT 1st Segment: Mammalian Cells Culture Overview
Mammalian Cells Culture Technology
Why Chosen Mammalian Cells???
From DNA to Protein- Transcription, Translation, Post translational modifications.
Type of cell Lines and Mode of Culturing
Current Cell Lines for Production
Products in The Market from Mammalian Cell Culture
Cell Banking & Cryopreservation
2nd Segment: Setting up a Cell Culture Laboratory
Basic Equipment
Aseptic work area: Biosafety Cabinet
Cell Culture Environment (in vitro): Media, supplements etc
Serum VS SFM
Standard cell culture techniques
Source of Contamination
Lab Briefing: Aseptic handling

3. Mammalian Cell Culture Overview
1st Segment
Mammalian Cell Culture Overview

4. Mammalian Cell Culture Technology
The use of mammalian cells culture for a wide range of biological and medical science technological application, such as:
Production of Biopharmaceutical Products
Erythropoetin, Interferon, Interleukins, tissue type Plasminogen activator, Monoclonal antibodies, vaccines etc.
2) Production of Gene Therapy Vectors
Retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses
3) Tissue Engineering, Cell Therapy and Organ Replacement
Artificial skin for severe burns, liver assist devices for hepatic failure, pancreatic islet of Langerhans devices for diabetes, etc.
4) In vitro substrates for pharmacological testing. Evaluation of effect
- reduce growth rate breakdown of membrane permeability
- tissue specificity of response ability to metabolize toxic compounds
- stimulated wound healing damage repair by use of artificially
- Genetic effects constructed tissue
- mutagenesis, i.e. interaction with DNA

5. Production of Therapeutics: Why Choose Mammalian Cells???
Mammalian cells are required for the correct post translational processing (including glycosylation) of biopharmaceutical protein products
Consistency, reproducibility and ease of scale up
Mammalian ell systems are the preferred “cell factories” for the production of complex molecules and antibodies for use as prophylactics, therapeutics or diagnostics.

6. From DNA to Protein (Translational & Transcription)
Copying of genetic information from DNA to RNA called Transcription
DNA is too large to leave the nucleus (double stranded), but RNA can leave the nucleus (single stranded).
TRANSLATION
Decoding of mRNA into a Protein
Transfer RNA (tRNA) carries amino acids from the cytoplasm to the ribosome.
Each tRNA codes for a different amino acid.
Amino acids are joined together to make a protein.

7. POST TRANSLATIONAL MODIFICATIONS (PTM)
What is PTM?
Chemical modification of a protein after its translation.
These modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis.
Glycosylation?
Major post- translational modifications, with significant effects on protein folding, conformation, distribution, stability and activity.
In correct Glycosylation:
- Most pharmaceutical relevant proteins are highly glycosylated
- incorrect glycosylated proteins may not be fully functional and/or might exhibit non- desired pharmacological behavior.

8. Glycosylation in different expression systems

9. Type of Cell Lines - Serial subculture of primary cells
- Initiated from normal, embryonic or malignant tissue by aseptic collection from animal host.
- Used in cellular metabolism &physiology, cell morphology and genetics.
- Considered primary until 1st subcultures
- Obtained directly from the organ of interest
- Serial subculture of primary cells
- Capable of continual growth, supplied with nutrients via periodic media change
- Can be stored in LN2 and revived for later use
- Utilized for the production of biopharmaceuticals, vaccines, gene therapy vectors or as an fusion partner with other cell types

10. Mode of Culturing (Adherent VS. Suspension)
There are two basic systems for growing cells in culture as, 1) Monolayers on articial substrate (i.e., adherent culture) or 2) free- floating (suspension culture).
Adherent Cell Culture
Suspension Cell Culture
Appropriate for most cell types, including primary cultures
Appropriate for cells adapted to suspension culture and a few other cell lines that are non-adhesive (e.g., hematopietic)
Required periodic passaging, but allows easy visual inspection under inverted microscope
Easier to passage, but required daily cell counts and viability determination to follow growth patterns; culture can be diluted to stimulate growth
Cells are dissociated enzymatically (e.g., TrypLETM Express, trypsin) or mechanically
Does not require enzymatic or mechanical dissociation
Growth is limited by surface area, which may limit product yields
Growth is limited by concentration of cells in the medium, which allows easy scale-up
Required tissue-culture treated vessel
Can be maintained in culture vessels that are not tissue-culture treated, but required agitation (i.e., shaking or stirring) for adequate gas exchange
Used for cytology, harvesting products continuously, and many research applications
Used for bulk protein production, batch harvesting , and many research applications.

11. Model of Culturing (Adherent VS. Suspension)
Adherent Cell Culture
Suspension cell Culture
Easily recognize by single cell
Free moving in Container
E.g: Spinner & Shake Flask
Strongly attached to the surface of Container
E.g: T-flask, Roller Bottle

12. Current Cell Lines for Prodcution
CHO Cells
Popular, robust cell line for the production of recombinant proteins i.e. Erythroooeitin, tissue plasminogen activator
Adaptable to serum free and suspension cell growth
Availability of mutant cell line deficient in DHFR gene, can be used to isolate recombinant clones and amplify expression levels.
VERO cells
Verde (French for green) and RenO (French for kidney
Epithelial morphology, non tumongenic, highly anchorage dependent and susceptible to infection by virus
Production for viral vaccines i.e. poliomyelitis and rabies
Sp2/0 Myelomas
Has been used as fusion partners for the production of antibody secreting hybridomas
Produced no immunoglobulin chains of its own. Therefore all antibody genes in the resulting hybridomas would arise from the lymphocyte fusion partners.

13. Cell lines used in Biopharmaceutical production
Origin
Featured
Use
MDCK
Dog/Kidney
Epithelial like
Veterinary vaccine
CHO-K1
Hamster/Ovary
Recombinant proteins
VERO
Green Monkey/Kidney
Fibroblast
Viral vaccines (poplio)
BHK-21
Hamster/Kidney
Recombinant proteins, Veterinary vaccines (clone B)
MRC-S
Human/Lung
Fibroblast, finite
Human vaccines
WI-38
Human embryonic/Lung
Namawala
Human/Lymphoid (Spleen)
Suspension
Interferon production
Mouse Myeloma
Spleen Sp2/0 and NS1
Fusion to makes hybridoma

14. Product from Mammalian Cells
Generic/ Trade Name
Company
Year
Treatment
Produced by Hybridoma Cell Culture (Mouse Antibody)
Muromomab/ Orthoclone3 OKT3®
Johnson &Johnson
1986
Acute kidney graft rejection in cardiac and liver transplant
Ibritumomab/ Zavalin ®
Biogen Idec
2002
Radioimmunotheraphy in non-Hodgkin lymphoma
Tositumomab/ Bexxar ®
Glaxo Smith Kline
2003
Produced by Cell Culture (Chimeric Antibody)
Rituximab/ Rituxan ®
MabThera ®
Biogen Idec/Genentech Inc
1997
Non-Hodgkins lymphoma, rheumatoid arthritis
Infliximab/ Remicade®
Centocor/ J&J
1998
Rheumatoid arthritis, Crohn’s disease, psoriasis
Centuximab/ Erbitus ®
Imclone Sys./Bristol-Myers
2004
Colorectal cancer
Poduced by Cell Culture (Recombinant Humanized Antibody)
Trastuzumab/Herceptin ®
Genentech Inc./Roche
Metastatic breast cancer
Gentuzumab/Mylotarg®
Wyeth Pharmaceuticals
2000
Acute myelotic leukemia
Bevacizumab/ Avastin ®
Genentech Inc
Metastatic rectal and colon cancer
Alexion Pharmaceuticals
2007
Paroxysmal nocturnal hemoglobinuria

15. Setting up a Cell Culture Laboratory
2nd Segment
Setting up a Cell Culture Laboratory

16. Criteria for Cell Culture Laboratory
Space allocated for the tissue culture should be one dedicated to tissue culture ONLY.
Minimize entry and exit
eg: by having freezer for medium and supplements storage, centrifuge etc
“air lock” space
- to help and ensure a “clean” tissue culture room
Ventilation: equipment placed at the same room
Maintained and clean routinely/daily.
Floors should be smooth and un-textured.
Clean benches: the dust-free assembly of sterile equipment or electronic devices.

17. Example Cell Culture Layout

18. Requirements for Tissue Culture Facilities
Minimum requirements
Desirable features
Useful additions
Sterile area, clean, quiet, and with no through traffic
Separate from animal house and microbiological labs
Preparation area
Wash up area (not necessarily within tissue culture laboratory, but at least adjacent to it)
Space for incubators(s)
Storage areas:
Liquid ambient, 4ºC, - 20º;
Glassware (shelving)
Plastics (shelving)
Small items (drawers)
Specialized equipment (slow turnover), cupboard(s)
Chemicals: ambient, 4º,- 20 º; share with liquids, but keep chemicals in sealed container over desiccant CO2 cylinders
Space for liquid N2 freezer(s)
Sink
Filtered air (air-conditioning)
Service bench adjacent to culture area
Separate prep room
Hot room with temperature recorder
Separate sterilizing room
Separate cylinder store
Piped CO2 and compressed air
Storeroom for bulk plastics
Quarantine room (could double as quarantine room)
Liquid N2 storage tank (~500L) and separate storeroom for nitrogen freezers
Microscope room
Darkroom
Vacuum line

19. Equipment for Tissue Culture Laboratory
Basic equipments
Nonessential, but beneficial
Useful additions
Laminar-flow hood (biohazard if for human cells)
Incubator humid CO2 incubator if using open
plates or dishes)
5% CO2 cylinder (for gassing cultures)
Liquid CO2 cylinders, without siphon (for CO2
incubator)
Balance
Sterilizer (autoclave, pressure cooker)
refrigerator
Freezer (for -20 º C storage)
Inverted microscope
Soaking bath or sink
Deep washing sink
Pipette cylinder(s)
Pipette washer
Still or water purifier
Bench centrifuge
Liquid N2 freezer (~35L, 1,500- 3,000 ampoules)
Liquid N2 storage Dewar (2.5L)
Slow-cooling device for ell freezing
Magnetic stirrer racks for suspension cultures
Hemocytometer
Cell counter
Peristaltic pump
Pipette(s)
PH meter
Sterilizing oven
Hot room
Temperature recorders on sterilizing oven and
autoclave and in hot room
Phase-contrast, fluorescence microscope
Pipette plunger
Pipette drier
Automatic dispenser
Trolley or carts
Drying oven(s), high and low temperature
Roller racks for roller bottle culture
Piped CO2 supply from cylinder store
Automatic changeover device on CO2 cylinders
Glassware washing
machine
Low-temperature (≤70ºC)
freezer
Conductivity meter
Osmometer
Polyethylene bag sealer (for packaging sterile items for long-term storage)
Computer for freezer
records and cell line
database
Colony counter
High-capacity centrifuge (6×1 L)
Digital camera and monitor
for inverted microscope(s)
Time-lapse video
equipment
Cell sizer (eg, Scharfe,
Coulter).
Portable temperature
recorder for checking
hot room or incubators
Plastics shredder/ sterilizer
Controlled-rate cooler for
cell freezing)
Fluorescence-activated cell
sorter
Confocal microscope
Microtiration plate
scintillation counter
Centrifugal elutriator
centrifuge and rotor

20. Critical Equipment (Biosafety Cabinet)
Should be properly set up and be located in an area that is restricted to cell culture that is free from doors, windows, other equipment, and with no through traffic.
Must be wiped clean before and after use.
Wipe the work surface with 70% ethanol before and during work, esp after any spillage.
Leave the BSC running at all time, turning it off only when they will not be used for extended periods of time.
HEPA filtered air.
UV light to kill bacteria also mutates human DNA, so it cannot be on when using the hood.

21. Biosafety Cabinet (Area Classification)
Offer signification levels of protection to laboratory personnel and to the environment when used with good microbiological technique.
But they do not provide cultures protection from contamination. They are similar in design and air flow characteristics to chemical fume hoods.
Used in general microbiological research with low and moderate risk agents.
Class II
Designed for work involving BSL-1,2, and 3 materials, and they also provide an aseptic environment necessary for cell culture experiments.
Used for handling potentially hazardous materials (e.g. primate- derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents.
Class III
Gas-tight, and they provide the highest attainable level of protection to personnel and the environment.
A class III biosafety cabinet is required for work involving known human pathogens and other BSL- 4materials.

22. Biosafety Cabinet (Area Classification)
Product protection (no personnel protection)
Not for biohazard agents or chemical fumes
Class I BSC: Personnel and Environment Protection
Class II & III BSC: Personnel, Product and Environment Protection
HEPA filters (not for chemical vapors)
Class I
Class II
Class III

23. Air-Flow Characteristics of Cell Culture Hoods
BSC: protect the working environment from dust and other airborn contaminants by maintaining a constant, unidirectional flow of HEPA- filtered air over the work area.
Different between laminar-flow chamber and biosafety cabinet is that air which comes out of the biosafety cabinet if filtered using HEPA filter (High Efficiency Particulate Air Filter), where in laminar-flow air blow out unfiltered so there is no protection for the laboratory worker.
The flow can be
Horizontal
blow parallel to the work surface
Provides protection to the culture (if the air flowing toward the user) or user (if the air is drawn in through the front of the cabinet by negative air pressure inside).
Vertical
blowing from the top of the cabinet onto the work surface
Protection significant protection to the user and the cell culture.

24. CRITICAL EQUIPMENT (CO2 Incubator)
Incubators
Should be large enough for your lab.
Have forced-air circulation
Temp control to within ± 0.2 ºC.
Stainless steal: easy cleaning, corrosion protection
Frequent cleaning of the incubator is a MUST
2 types of incubator:
- Dry incubators
- Humid CO2 incubators

25. CRITICAL EQUIPMENT (Cryogenic Storage)
Two main types of liquid-nitrogen storage systems, vapor phase and liquid phase.
Vapor phase- minimize the risk of explosion with cryostorage tubes, and are required for storing biohazardous materials.
Liquid phase- usually have longer static holding times, more economical.
Narrow-necked containers- slower nitrogen evaporation rate, economical.
Wide-necked containers- allow easier access and have a larger storage capacity.
Vapor phase
Liquid Phase

26. Cell Culture Environment (in Vitro) What do cells need to grow?
Culture media/ basal medium (Nutrients sources, growth factors).
Environment (CO2, temperature 37 ºC, humidity).
Sterility (aseptic technique, antibiotics and antimycotics, Mycoplasma tested).
The three basic classes of media are:
- basal media
must be further supplemented with serum.
Maintain pH(pH 7.4) and osmolarity ( mOsm/L).
Others components: inorganic salts, buffer, glucose etc.
- reduced- serum media
- serum- free media
(which differ in their equipment for supplementation with serum).

27. Supplements Unstable in liquid media- added as a supplement.
Energy source (citric acid cycle), used in protein synthesis.
Essential amino acid (not synthesized by the cell).
L- glutamine
May reduce metabolic burden on cells.
Energy source, used in protein synthesis
Usually added to basic media compositions.
Non- essential amino acids (NEAA)
Maintenance of differentiation
Uptake of amino acids
Stimulate glucose transport and utilization
Growth Factors and Hormones (e.g.: insulin)
Preferably avoided in long term culture.
Cells can become antibiotics resistant- changing phenotype
Reduce the risk of bacterial and fungal contamination
Penicillin, streptomycin, gentamicin, amphotericin B
Antibiotics and Antimycotics

28. Types of Cell Culture Media
Serum Based Media
Natural media, useful for a wide range of animal cell culture.
Immediate survival of cells, prolonged survival, for indefinite growth and also for specialized function.
Serum Free Media
Design to grow a specific cell type or perform a specific application in the absence of serum.
May contain serum constituent/ substitute thereof
Animal Free Media
Similar to serum-free except that the components are derived from non-animal sources.
Recombinant proteins replace native proteins and the nutrients are obtained from synthetic, plant or microbial sources.
Protein Free Media
Defined as devoid of protein, although few formulations of these media can be 100% protein-free without loss of function.
Low protein where minimal quantities of small mass proteins are employed.
Chemically defined Media
Basal formulation which may also be protein-free and is comprised solely of biochemically-defined low molecular weight constituents.

29. Serum VS. Serum Free Media
Added to media as source for growing cells.
Disadvantages:
- Costly
- Unwanted effects (stimulation or inhibition of growth)
- Regulatory issues
- Specificity: might contain of variable composition
Serum Free
Replacing serum with appropriate nutritional and hormonal formulations
Advantages:
- Simplified and better defined composition.
Ability to make medium selective for specific cell types (CHO, other recombinant cell types)
Reduced degree of contaminants.

30. Serum Free Media ADVANTAGES
Serum contains hormones and growth factors, which play a major role in stimulating cell growth and function.
Serum helps in attachment of the cells and as spreading factors.
Serum minimized mechanical damage and also damage caused by viscosity.
Serum also acts as natural buffering agent and helps in maintaining the pH of the culture media.
DISADVANTAGES
May contain some of the growth inhibiting factors, will inhibits the cultured cell growth.
Got high risk of contamination with virus, fungi and mycoplasma.
Serum availability is restricted as they extracted from cattle.
Serum may interfere with the purification of cell culture products, i.e pharmaceutical compounds.

31. Source of Contamination
A cell culture contaminant can be defined as some element in the culture system that is undesirable because of its possible adverse effects on either the system or its use.
Chemical Contamination
Media
Incubator
Serum
Water
Biological Contamination
Bacteria & Yeast
Viruses
Mycoplasmas
Cross Contaminations
Molds

32. Standard Cell Culture Techniques
Thawing/ Seed
Subculture
is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain.
To allow cells to remain in exponential growth
Cryopreserve
To assure the continuous supply
To provide a secure cell stock
To protect against accidental loss and genetic and phenotypic instability.
‘Mr. Frosty
Used to freeze cells.

33. LAB BRIEFING Aseptic techniques, sterile handling and sterile consumables overview
Why is important of Aseptic technique?
To prevent microbial contamination of cultures.
To prevent cross- contamination of cell cultures as well.
Major areas in handling aseptic techniques:
Sterile Work Area: BSC(II), Incubator.
Good Personal Hygiene- wear PPE.
Sterile Handling.
Sterile Reagents and Media.

34. PPE & Gowning procedure for entering the cell culture laboratory
Put shoe covers on over designated laboratory shoes.
Clean hands with the hand sanitizer provided.
Put on a NEW bouffant hair cap, ensuring all loose hair is neatly tucked in.
Put a NEW face mask on.
Put on the designated tissue culture laboratory coat, ensuring all fastenings are secure (front and cuffs).
Put on NEW appropriately sized gloves, tucking laboratory coat sleeves into the gloves.
Goggles are optional and are available in the tissue culture laboratory.

35. Degowning procedure for entering the cell culture laboratory
Take off your gloves and clean your hands with the hand sanitizer provided. Remove your bouffant hair cap.
Remove your face mask.
Take off your tissue culture laboratory coat and put it on the hanger provided.
Take off the top layer of shoes cover, one side at a time, and place each foot on the main lab side (over the boundary marked on the floor) after taking off each shoe cover.

36. ASEPTIC HANDLING
Always wipe your hands and your work area with 70% ethanol.
Wipe the outside containers, flasks and dishes with 70% ethanol before placing them in the cell culture hood. Use only sterile glassware or other equipment.
Arrange your work area so that you have easy access to all items without having to reach over one to get at another.
Work within your range of vision.
Use sterile glass or disposable plastic pipettes to work with liquids, and use each pipette only once to avoid cross contamination. Do not unwrap sterile pipettes until they are to be used.
Always keep cap the bottles and flask after use to prevent microorganisms from gaining entry.
Bottles of media and other reagents SHOULD NOT share with other people or use for different cell lines.

37. 8. Never uncover a sterile flask, bottle, petri dish, etc
8. Never uncover a sterile flask, bottle, petri dish, etc. until the instant you are ready to use it and never leave it open to the environment. Return the cover as soon as you finished. 9. If you remove the cap or cover, and have to put it down on the work surface place the cap with the opening facing down. 10. Wise up any spillage immediately with 70% ethanol. 11. Be careful not to talk, sing or whistle when you are performing sterile procedure. 12. Perform your experiments as rapidly as possible to minimize contamination. 13. Remove everything once your finished your work and wipe your work area with 70% ethanol. 14. Decontaminate all potentially infectious materials before disposal.

38. Sterile Reagents, media and consumables

39. Waste Disposal Management
Waste Storage Room