Animal Cell Culture Chapter Notes | Biotechnology for Class 12 - NEET PDF Download

Introduction

• Animal cell culture involves the in vitro maintenance and proliferation of animal cells outside the living organism, requiring appropriate nutrients and growth conditions.
• Cell culture is the process of growing cells under controlled laboratory conditions, performed in vitro as opposed to in vivo (within a living organism).
• It includes isolating cells from animal tissues, often through surgical removal of tissues or organs, and placing them in a suitable environment (media) to support survival and proliferation.
• A clone is a homogenous population of cells derived from a single parental cell, ensuring all cells are genetically identical.
• Animal cells grow relatively slowly, typically requiring 18 to 24 hours to divide, making them susceptible to contamination by faster-growing bacteria.
• Optimal cell growth requires a regulated temperature, a proper substrate for cell attachment, an appropriate growth medium, and an incubator maintaining correct pH and osmolality.
• Cell culture is a dominant tool in life sciences, used to study cell proliferation, differentiation, and product formation under controlled conditions.
• It has applications in molecular genetics, immunological analysis, surgery, bioengineering, and the pharmaceutical industry.

Historical Perspective

• Animal cell culture became a routine laboratory technique in the 1950s after George Gey established the HeLa cell line from Henrietta Lacks’ cervical cancer cells.
• The HeLa cell line, the first human cell line, led to significant medical discoveries, including the development of the polio vaccine.
• The need for large-scale cell culture arose with the demand for viral vaccines, driving advancements in cell culture technologies.
• In 1882, Sydney Ringer developed a balanced salt solution mimicking body fluids, used to keep frog hearts alive post-dissection.
• In 1885, Roux maintained the medullary plate of a chick embryo in warm saline, an early step in tissue culture.
• In 1903, Jolly demonstrated in vitro cell survival and division of salamander leucocytes.
• In 1907, Ross Harrison showed frog embryo nerve fiber growth in vitro, a significant milestone in cell culture.
• In 1911, Lewis and Lewis cultured connective tissue cells for extended periods and demonstrated heart muscle tissue contractility for 2–3 months using liquid media with sea water, serum, embryo extracts, salts, and peptides.
• In 1912, Alexis Carrel introduced aseptic techniques to tissue culture, using trypsin, embryo extracts, and animal serum.
• In 1913, Rous and Jones introduced antibiotics (penicillin/streptomycin) to control contamination.
• In 1916, laminar air-flow cabinets were used to maintain sterile conditions.
• In the 1940s, trypsinization was employed to produce homogenous cell types, and tissue culture media were developed.
• In the 1940s–50s, Katherine Sanford and others cloned mouse L-cells, showing tumor cells could form continuous cell lines and using non-malignant rodent cells to study carcinogens and viruses.
• In 1948, Margaret and George Gey observed contact inhibition among fibroblasts, marking the start of quantitative cell culture experimentation.
• In 1952, Abercrombie and Heaysman studied polio virus in human E-cells, aiding polio vaccine production.
• In 1954, Enders and others developed human cell lines for human and veterinary vaccine production.
• In 1955, Hayflick and Moorhead described the finite lifespan of normal human diploid cells.
• In 1961, methods for maintaining differentiated cells of tumor origin were published.
• In 1962, Harry Eagle developed defined media, improving culture consistency.
• In 1962, Buonassisi and others studied the differentiation of normal myoblasts in vitro.
• In 1964, Littlefield introduced HAT selection for hybridoma technology.
• In 1968, David Yaffe worked with human fetal lung fibroblasts.
• In 1975, Kohler and Milstein developed the first hybridoma capable of producing a monoclonal antibody, revolutionizing immunological research.
• In 1976, the development of serum-free media accelerated, reducing reliance on animal-derived components.

Culture Media

• Selecting an appropriate growth medium is critical for successful in vitro animal cell culture, depending on the cell type and culture purpose (e.g., growth, differentiation, or product formation).
• A typical culture medium contains vitamins, amino acids, glucose, inorganic salts, serum (as a source of growth factors), and hormones to support cell survival, proliferation, and function.
• The medium maintains pH and osmolality, ensuring optimal conditions for cell growth.
• Media are categorized into natural media (containing biological substances like plasma, serum, or tissue extracts) and artificial/synthetic media (basal medium with supplements like hormones, growth factors, or serum).
• Serum, often fetal bovine serum, is a key component, providing amino acids, proteins, vitamins, carbohydrates, lipids, hormones, and growth factors.
• Serum includes binding proteins (e.g., albumin, transferrin) that transport molecules into cells and adhesion factors that help cells adhere to the substrate before division.
• Media supplements, such as hormones, growth factors, and signaling substances, are added to basal media to support proliferation and maintain normal cell metabolism.
• Examples of growth factors include nerve growth factor, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, platelet-derived growth factor, and transforming growth factor, which enhance cellular proliferation.
• Natural media include coagulants/clots (plasma, serum, fibrinogen), tissue extracts (e.g., chicken embryos, liver, spleen, bone marrow), and biological fluids (e.g., plasma, serum, lymph, amniotic fluid, pleural fluid).
• Synthetic media include serum-containing media (using human, bovine, or equine serum), serum-free media (using crude protein fractions like bovine serum albumin or globulins), xeno-free media (using human-source components like human serum albumin), protein-free media (using peptide fractions), and chemically defined media (basal media with balanced salt solutions).
• Chemically defined media contain inorganic salts to maintain physiological pH and osmotic pressure, acting as cofactors in enzyme reactions and aiding cell attachment.
• Examples of complex synthetic media include Eagle’s Minimum Essential Medium (EMEM), Dulbecco’s Minimal Essential Medium (DMEM), Roswell Park Memorial Institute (RPMI-1640), and Ham’s Nutrient Mixtures (Ham’s F-12).
• Disadvantages of serum include insufficient cell-specific growth factors, potential cytotoxic compounds, growth-inhibiting factors, and risks of contamination by viruses, fungi, or mycoplasma.
• Serum may interfere with the purification of cell culture products, such as pharmaceutical compounds, requiring additional isolation steps.
• Antibiotics like penicillin and streptomycin are commonly added to control bacterial and fungal contamination, though not essential for cell growth.
• Disadvantages of natural media include poor reproducibility and reduced uniformity due to unknown exact compositions.
• Serum quality varies between batches and deteriorates within a year, requiring fresh testing for each batch.
• Serum-free media allow selective medium formulation for specific cell types, as each cell type has unique requirements.

Physical Environment for Culturing Animal Cells

• In vitro animal cell culture requires a simulated microenvironment with appropriate nutritional, physical, and hormonal conditions for optimal cell growth.
• Key factors include controlling temperature, osmolality, pH, gaseous requirements, providing a supporting surface, and protecting cells from physical, chemical, and mechanical stresses.
• Mammalian cells are typically cultured at 37°C in CO₂ incubators, mimicking human body temperature, as most cells are derived from warm-blooded animals.
• Osmolality affects cell growth and membrane integrity; deviations from the cell’s internal osmotic pressure cause cells to swell or shrink.
• Glucose, salts, and amino acids contribute to medium osmolality, with commercial media formulated to approximately 300 mOsmol, measurable by an osmometer.
• pH regulation is achieved through buffering systems, either natural (using gaseous CO₂ balanced with CO₂/HCO₃⁻ in the medium) or chemical (using HEPES).
• In a natural buffering system, 5–10% CO₂ is maintained in the incubator, and pyruvate in the medium increases endogenous CO₂ production, reducing reliance on external CO₂.
• Natural buffering systems are low-cost and non-toxic, with pH stabilized by bicarbonate reacting with excess H⁺ ions to form carbonic acid (Le Chatelier’s principle).
• HEPES, a zwitterionic buffer, provides superior buffering capacity at pH 7.2–7.4, eliminating the need for a controlled gaseous atmosphere but is expensive and toxic at high concentrations for some cells.
• Phenol red, a pH indicator in commercial media, changes color with pH: yellow (acidic), bright red (optimal pH 7.4), or pink (alkaline), reflecting metabolic activity during cell growth.

Equipment Used for Cell Culture

• Cell cultures are maintained in flasks, Petri dishes, or multi-well plates, incubated at 37°C, 95% humidity, and 5% CO₂ for mammalian cells, with daily checks for medium color, cell morphology, and density.
• Culture conditions vary by cell type, and deviations can alter cell phenotypes.
• Essential equipment includes laminar flow hoods, CO₂ incubators, inverted microscopes, autoclaves, and centrifuges.
• Laminar flow hoods (biosafety cabinets) ensure aseptic conditions by filtering air through HEPA filters, removing particulates, microbes, and contaminants.
• Air in laminar hoods is drawn under negative pressure, passes through a pre-filter to trap fungi, bacteria, and dust, and is then fed through HEPA filters into the cabinet under positive pressure to prevent contaminated air inflow.
• Vertical laminar flow hoods direct sterilized air downward, safer as air does not blow toward the operator, unlike horizontal flow hoods, which are less popular due to lack of operator protection.
• CO₂ incubators maintain sterility, constant temperature (37°C), 5–10% CO₂, and ~95% humidity, with external CO₂ cylinders piped in to avoid contamination.
• The medium in CO₂ incubators is buffered with sodium bicarbonate/carbonic acid to strictly maintain pH.
• Inverted microscopes with phase-contrast optics and photographic capabilities monitor cell morphology, granularity, spreading, membrane blebbing, multinucleation, vacuolation, and stress signs.
• Regular microscope checks detect microbial contamination early, preventing loss of valuable cultures and ensuring reliable experimental results.
• A long or extra-long working distance condenser is recommended for viewing flasks and roller bottles, with a 20x magnification objective sufficient for most observations.
• Low-power, wide-field objectives are useful for scanning culture colonies, as higher magnification may lack sufficient depth of field for sharp imaging.

Types of Animal Cell Cultures and Cell Lines

• Animal cell cultures are classified as primary or secondary cell cultures and cell lines, further categorized by lifespan as finite or continuous cell lines.
• Primary cell cultures are derived directly from host tissue, dissociated mechanically or enzymatically (e.g., with trypsin or collagenase), and grown in suitable media.
• Tissues for primary cultures are dissected under sterile conditions, chopped, or disaggregated, with cells either adhering to a dish or growing from tissue fragments (explants).
• Primary cultures are heterogeneous, resembling parental cells, and can grow as adherent monolayers or in suspension.
• Secondary cell cultures are obtained by sub-culturing (passaging) primary cultures, transferring a portion of cells to a new vessel with fresh medium for continued growth.
• Sub-culturing leads to secondary cell lines, where cells with the highest growth capacity dominate, resulting in genotypic and phenotypic uniformity.
• Repeated sub-culturing may cause cells to differ from the original population.
• Finite cell lines have a limited number of divisions, undergo senescence, and stop proliferating; they are derived from primary cultures of normal cells.
• Continuous cell lines arise when finite cell line cells undergo transformation (spontaneous, chemical, or viral), gaining indefinite division capacity and becoming immortal and tumorigenic.
• Continuous cell lines are less adherent, can grow in suspension, are fast-growing, less nutrient-demanding, and can reach higher cell densities.
• Adherent cells are anchorage-dependent, proliferating as monolayers on a solid or semi-solid substrate, typically derived from immobile connective tissues (e.g., fibroblasts, epithelial cells).
• Monolayer cultures form a single cell layer, suitable for direct transfer to coverslips for microscopic examination, and most continuous cell lines grow as monolayers.
• Suspension cells (anchorage-independent) float in the medium, derived from hematopoietic stem cells (e.g., blood, spleen, bone marrow) or tumor cells, growing faster and requiring less frequent medium replacement.
• Sub-culturing (passaging) involves detaching adherent cells using enzymes (e.g., trypsin, trypsin + EDTA) or mechanical methods (e.g., cell scrapers, shaking, pipetting) and transferring them to fresh media after centrifugation.
• Passaging prevents overcrowding, which can lead to cell death, by splitting cells from one flask into multiple flasks with fresh medium.
• A passage number indicates how many times a cell line has been sub-cultured.
• Cryopreservation stores cells at –180 to –196°C in liquid nitrogen to minimize ice crystal formation, using cryoprotective agents like glycerol or DMSO (typically 90% serum, 10% DMSO).
• Freezing can damage cells due to ice crystals, electrolyte changes, dehydration, or pH shifts, so cells are cooled slowly to –80°C to allow water to exit before freezing.
• Healthy cells in the log phase are preferred for freezing, with medium replaced 24 hours prior.
• Frozen cells are thawed rapidly in a 37°C water bath with moderate shaking to minimize ice crystal damage, then transferred to a culture vessel with suitable media.

Cell Viability Determination

• Cell viability measurement determines the proportion of living cells in a culture, crucial for assessing culture health and experimental outcomes.
• Viability assays evaluate the effects of pesticides, insecticides, toxins, or drugs and are performed regularly to monitor cell status.
• Dye exclusion viability assays use stains (e.g., trypan blue, propidium iodide, 7-aminoactinomycin D, acridine orange) that enter cells with compromised membranes, indicating cell death.
• Live cells exclude the dye, while dead cells internalize it, staining the nucleus due to DNA intercalation, making it a dye-exclusion assay.
• Metabolic viability assays measure biochemical reactions, such as enzyme activity, using absorbance, fluorescence, or luminescence methods.
• The MTT assay uses a yellow water-soluble salt, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), reduced by mitochondrial dehydrogenase in live cells to purple insoluble formazan crystals.
• Formazan is dissolved in DMSO, and its amount, proportional to cell number, is measured, as dehydrogenase content is consistent for specific cell types.
• Metabolic assay results show color intensity directly proportional to the number of viable cells.

Advantages of Animal Cell Culture

• Allows growth in a controlled physico-chemical environment, ensuring consistent conditions.
• Produces a homogenous genetic population, ideal for genetic and biochemical studies.
• Provides adequate cell numbers for chemical and pharmaceutical studies.
• Facilitates easy production of biopharmaceuticals, such as therapeutic proteins.
• Does not require ethical clearance, unlike in vivo animal studies.
• Limitations include high sensitivity to small changes, which can reduce productivity, and potential failure to fully represent in vivo phenotypes or genotypes.

Applications of Animal Cell Culture

• Serves as a model system to study interactions between cells and disease-causing agents or drugs.
• Provides a convenient and economic tool for virus research, enabling study of viral replication and effects.
• Enables large-scale vaccine production, critical for public health.
• Acts as a production platform for medically important pharmaceutical proteins, such as follicle-stimulating hormone (FSH) for infertility, human growth hormone (HGH) for growth deficiency, erythropoietin (EPO) for anemia, factor VIII for hemophilia A, factor IX for hemophilia B, interleukin-2 (IL-2) for cancer therapy, tissue plasminogen activator (tPA) for stroke, and monoclonal antibodies (mAbs) for cancer therapy and diagnostics.
• Erythropoietin (EPO), a glycoprotein hormone, is produced in Chinese Hamster Ovary (CHO) cells to treat anemia caused by chemotherapy, AIDS, or chronic renal failure, stimulating red blood cell production and wound healing.
• Recombinant human EPO (r-HuEPO) reduces the need for blood transfusions, minimizing donor requirements and transfusion-associated disease risks.
• Factor VIII, a glycoprotein produced in CHO cells, treats hemophilia A, an inherited disorder causing deficient blood clotting.
• Factor IX, produced in CHO cells, treats hemophilia B (Christmas disease), a bleeding disorder due to factor IX deficiency.
• Tissue plasminogen activator (tPA), a serine protease produced in CHO cells, converts plasminogen to plasmin to dissolve blood clots, used for heart attack and stroke patients.
• Muromonab-CD3 (OKT-3), a murine monoclonal antibody targeting the CD3 receptor, is used as an immunosuppressant to reverse acute organ transplant rejection (e.g., kidney, heart, liver) by blocking T-cell function and promoting T-cell elimination via phagocytosis.
• OKT-3 was the first monoclonal antibody approved for clinical treatment of acute rejection, with T-cell function typically returning within a week post-therapy.
• Trastuzumab (Herceptin), a monoclonal antibody, treats early-stage HER2-positive breast cancer by binding to overexpressed HER2 receptors, blocking growth signals, and impairing cancer cell proliferation.
• Supports research and development of drugs, improving health and quality of life for patients with cancer, genetic disorders, and other diseases.