Experimental Pharmacology

Experimental pharmacology: a brief history of cell and tissue culture systems

Cell culture in vitro

• 1885: Roux maintained embryonic chick cells alive in saline solution for short lengths of time.
• 1912: Alexis Carrel cultured connective tissue and showed heart muscle tissue contractility over 2-3 months.
• 1943: Earle et al. produced continuous rat cell line.
• 1962: Buonassisi et al. published methods for maintaining differentiated cells (of tumor origin).
• 1970s: Gordon Sato et al. published the specific growth factor and media requirements for many cell types.
• 1979: Bottenstein and Sato defined a serum-free medium for neural cells.
• 1980 to date: Tissue culture becomes more of a widely accepted research tool.

Why do we need cell culture systems?

• Studying in detail the basic molecular mechanism of pathology.
• Testing the new pharmacological target before moving to in vivo models of pathologies.
• Stem cells research: possible clinical intervention of tissue regeneration.
• Reconstitute tissue in vitro for clinical intervention.

Cell or tissue from a resected tissue grows in vitro, forming a primary culture. Then, a sub-culture is extracted to form a secondary culture and obtain a cell line, which could be immortalized to obtain a transformed cell line.

Primary cultures

• Derived directly from animal tissue.
• Embryo or adult? Normal or neoplastic?
• Cultured either as tissue explants or single cells.
• Initially heterogeneous – become overpopulated with fibroblasts.
• Finite life span in vitro.
• Retain differentiated phenotype.
• Mainly anchorage dependent.
• Exhibit contact inhibition.

They can be obtained by mechanical or enzymatic dissociation of tissue fragments explanted from fresh organs. The explants can derive from adult tissue or embryo tissue (cells grow a little faster). Best example: human umbilical vein endothelial cells (HUVEC).

Some cells can survive only for a few days without duplication → red cells. Some can survive for weeks without duplication → neurons, hepatocytes. Others can grow and expand → skin fibroblasts, endothelial cells, smooth muscle cells.

Secondary cultures

• Derived from a primary cell culture.
• Isolated by selection or cloning.
• Becoming a more homogeneous cell population.
• Finite life span in vitro.
• Retain differentiated phenotype.
• Mainly anchorage dependent.
• Exhibit contact inhibition.

Continuous cultures

• Derived from a primary or secondary culture.
• Immortalized: spontaneously (natural genetic mutation) or by transformation vectors (viruses, plasmids).
• Serially propagated in culture showing an increased growth rate.
• Homogeneous cell population.
• Loss of anchorage dependency and contact inhibition.
• Infinite life span in vitro.
• Differentiated phenotype:
  • Retained to some degree in cancer derived cell lines.
  • Very little retained with transformed cell lines.
  • Genetically unstable.

Cell morphologies

Fibroblastic, Epithelial, Endothelial, Neuronal.

Cells can grow in suspension or in adhesion. Monocytes (THP-1) grow in suspension, but when treated with PMA they differentiate into macrophages, that grow in adhesion. PMA is a phorbol ester that activates the PKC changing the gene expression and cell phenotype.

Stem cell types

They can duplicate and regenerate or differentiate into different cells like neurons, adipocytes, etc.

• HSCs (hematopoietic stem cells): adult stem cells found in bone marrow and blood. Capable of producing all of the cells that make up the blood and the immune system.
• MSCs (mesenchymal stem cells): adult stem cells found in several places in the body, including the bone marrow, skin, and fat tissue. They produce cells which help other stem cells function properly.
• NSCs (neural stem cells): specialized stem cells responsible for repairing nerve-insulating myelin in the brain. These can be derived from other types of stem cells such as mesenchymal cells.
• hESCs (human embryonic stem cells): stem cells derived from donated embryos. They can naturally produce every type of cell in the body. One concern about their potential therapeutic use is that they have been found to cause tumors.
• iPSCs (induced pluripotent stem cells): engineered from adult cells to produce many types of cells. One concern about their potential therapeutic use is that they have been found to cause tumors.

The ideal is to take somatic cells from the skin fibroblasts of a patient, they grow in culture, expanding themselves and then some pluripotency factors are added: SOX2, KLF4, c-MYC, and OCT4, in order to obtain pluripotent stem cells. The cells carry all the genotype of the patient, so they can differentiate in different types of cells, so they can be used to regenerate some organs, to study human diseases difficult to study and to do drug screening to treat them.

Example: iPS can differentiate into cardiomyocytes, that will be later injected into a damaged heart. This approach is in clinical trial for humans. Some regeneration is visible, but these cells induce arrhythmia. A similar therapy is tested for the liver.

Cell culture environment

To grow, cells need:

• Substrate or liquid: Chemically modified plastic or coated with EMC proteins (collagen, elastin…).
  • Suspension culture.
• Nutrients (culture media).
• Environment: CO₂ 5%, temperature 37°C, humidity 95% (needed to not let culture media evaporate). Oxygen tension maintained at atmospheric but can be varied.
• Sterility: aseptic technique, antibiotics, and anti-mycotics (Mycoplasma tested).

Basal media maintains pH and osmolarity (260-320 mOsm/L) and it provides nutrients and energy source. Components:

• Inorganic salts: maintain osmolarity, regulate membrane potential (Na⁺, K⁺, Ca²⁺). Ions for cell attachment and enzyme cofactors.
• pH Indicator: Phenol Red, optimum cell growth approx. pH 7.4 (red). It turns into violet with more basic conditions or into orange/yellow in more acidic conditions. HCO₃^- + H⁺ → CO₂ + H₂O optimum at eq.
• Buffers (Bicarbonate and HEPES)
  • Bicarbonate: media requires CO₂ atmosphere.
  • HEPES is a strong chemical buffer, range pH 7.2 – 7.6, it does not require CO₂.
• Keto acids (oxaloacetate and pyruvate): intermediate in glycolysis/Krebs cycle. Keto acids added to the media as an additional energy source. Maintain maximum cell metabolism.
• Carbohydrates: glucose and galactose, energy source. Low (1 g/L) and high (4.5 g/L) concentrations of sugars in basal media.
• Vitamins: precursors for numerous co-factors, B group is necessary for cell growth and proliferation. Common vitamins found in basal media are riboflavin, thiamine, and biotin.
• Trace Elements: zinc, copper, selenium, and tricarboxylic acid intermediates.

Supplements

• L-glutamine (5 ml): essential amino acid, not synthesized by the cell. Energy source (citric acid cycle), used in protein synthesis. Unstable in liquid media → added as a supplement.
• Non-essential amino acids (NEAA) (5 ml): usually added to basic media compositions. Energy source, used in protein synthesis. May reduce metabolic burden on cells.
• Growth factors and hormones (like insulin). Stimulate glucose transport and utilization. Uptake of amino acids and maintenance of differentiation.
• Antibiotics and antimycotics (5 ml): penicillin, streptomycin, gentamicin, amphotericin B. Reduce the risk of bacterial and fungal contamination. Cells can become antibiotic-resistant → changing phenotype. Preferably avoided in long term culture.

Fetal Calf/Bovine Serum (FCS & FBS) (50 ml):

• Growth factors and hormones.
• Aids cell attachment.
• Binds and neutralizes toxins.
• Long history of use.
• Infectious agents (prions).
• Variable composition.
• Expensive.
• Regulatory issues (to minimize risk).
• It arrives completely frozen (-20°C) so it needs to be defrosted and to keep inactive → heat inactivation (56°C for 30 mins). Why? Destruction of complement and immunoglobulins and of some viruses (also gamma-irradiated serum). Care! Overdoing it can damage growth factors, hormones, and vitamins and affect cell growth. When not put in a culture, cells can be stored in freezing vials and kept in liquid nitrogen (-196°C, cryopreservation).

Usually, cells keep growing till they reach the maximum confluency and form a layer of cells, while tumor cells keep growing also after this step, forming different layers.

Cell culture incubator

It requires a controlled atmosphere with high humidity and super controlled CO₂ tension. The incubator should be large enough, probably 50-200 liter, have forced air circulation, temperature control, and a safety thermostat that cuts out if the incubator overheats. It should be stainless steel and easily cleaned. A double cabinet, one above the other, independently regulated, is preferable to one large cabinet.

Incubators are supplied either with a heated water jacket as a method for distributing the heat evenly around the cabinet or with surface heater elements for heating.

CO₂ incubators are more expensive, but they’re easier to use and superior in the control of CO₂ tension and temperature. A controlled atmosphere is achieved by using a humidifying tray and controlling the CO₂ tension with a CO₂-monitoring device, which draws air from the incubator into a sample chamber, determines the concentration of CO₂, and injects pure CO₂ into the incubator to make up any deficiency.

Air is circulated around the incubator by natural convection or by using a fan to keep both the CO₂ level and the temperature uniform. Dry, heated wall incubators also encourage less fungal contamination on the walls, as the walls tend to remain dry, even at high relative humidity.

Biohazard hood

The air is completely filtered, so the atmosphere is sterile, but the materials outside the hood aren’t. So sterility starts from a certain point of the hood, not close to the open.

Filters utilized are HEPA filters (High Efficiency Particulate Air filters): replaceable extended-media dry-type filters in a rigid frame having a minimum particle collective efficiency of:

• 99.97 percent for a 0.3 micron particle (standard grade).
• 99.90 percent for a 0.3 micron particle (low grade).
• 99.99 percent for a 0.3 micron particle (high grade).

And a maximum clean filter pressure drop of 2.54 cm (1") water gauge when tested at rated air flow capacity.

Contamination

Cell culture contaminants come in two types:

• Chemicals: difficult to detect; caused by endotoxins, plasticizers, metal ions or traces of disinfectants that are invisible.
• Microorganisms: a major problem in tissue culture, even using penicillin, gentamycin and/or streptomycin. Bacteria, mycoplasma, yeast, and fungal spores may be introduced via the operator, the atmosphere, work surfaces, solutions, and many other sources. Very difficult to get rid of all of them.

Yeast, mold, bacteria, and mycoplasmas can be contaminants.

Contaminants compete for nutrients with host cells, secrete acidic or alkaline bio-products that cease the growth of the host cells and degraded arginine and purine inhibits the synthesis of histone and nucleic acid. They also produce H₂O₂ which is directly toxic to cells.

For best results in tissue culture, we want to work to keep microbial (bacteria, yeast, and molds) contamination to a minimum. Guidelines to follow:

• Work in a culture hood set aside for tissue culture purposes.
• Most have filtered air that blows across the surface to keep microbes from settling in the hood. Turn off the UV/antimicrobial light and turn on the hood 30 minutes prior to entering the hood.
• Wear short sleeves or roll your sleeves up. Tie long hair back and remove rings and watches.
• Wash hands with soap and water before beginning the procedure and rewash if you touch anything that is not sterile or within the hood.
• Spray down your hands, work surface, and anything that will go into the hood with 70% ethanol. Re-wipe at intervals if you are working for a long time in the hood. This will reduce the numbers of bacteria and mold considerably.
• Do not breathe directly into your cultures, bottles of media, etc. This also means to keep talking to a minimum. No singing or chewing gum.
• Work as quickly as you can within limits of your coordination. Also, keep bottles and flasks closed when you are not working with them. Avoid passing your arm or hand over an open bottle.
• Use only sterilized pipettes, plates, flasks, and bottles in the hood for procedures. Take special precautions with the sterile pipette. Remove them from the package just before use. Make certain to set up the numbers on the pipette so that they face you.
• Never mouth-pipette, use the pipetting aid.

Sterilization (in microbiology)

To completely remove all kinds of microbes (bacteria, mycobacteria, viruses, and fungi) by physical or chemical methods. Effective to kill bacterium spores. Sterilant: material or method used to remove or kill all microbes.

Methods

Physical sterilants

• Steam under pressure: 121°C or 132°C for various time intervals. Sterilization stays 20-30 minutes.
• Dry heat: simple oven, 1 hour at 171°C, 2 hours at 160°C, or 16 hours at 121°C.
• Filtration: for small amounts of solution, with HEPA filters with 0.22 (nothing passes) -0.45 (some viruses pass) μm pore size.
• UV radiation: variable exposure to 254 nm wavelength.
• Ionizing radiation: variable exposure to microwave or gamma radiation.

Gas vapor sterilants

• Ethylene oxide.
• Formaldehyde vapor: used in labs when the filter is replaced.
• Hydrogen peroxide vapor.
• Plasma gas: highly ionized hydrogen peroxide gas.
• Chlorine dioxide gas: variable.

Chemical sterilants

• Peracetic acid: 0.2%.
• Glutaraldehyde: 2%.

Pros and cons

• Steam and Dry Heat: the most common methods for most materials.
  • Cons: Not good for heat-sensitive, toxic or volatile chemicals.
• Filtration: removes bacteria and fungi from air or solutions, like HEPA filters.
  • Cons: Unable to remove viruses and some small bacteria (mycoplasma).
• Ethylene oxide: the most common gas vapor sterilant.
  • Cons: Flammable and explosive, potential carcinogenic.
• Formaldehyde gas: carcinogenic.

An autoclave, a simple bench-top autoclave that generates 100 kPa (1 atm, 15 lb/in.²), may be sufficient, but a larger model with a timer and a choice of pre-sterilization and post-sterilization evacuation and temperature recording gives more capacity and greater flexibility in use. Offers the opportunity to comply with good laboratory practice (GLP).

Chemical monitoring involves the use of heat-sensitive chemicals that change color when they are exposed to certain conditions. Process indicators are placed on the outside of the instrument packages before sterilization. They respond to heat only.

In vitro assay

Tumor progression

Starts with the mutation of a particular cell and it grows uncontrollably. Some of the cells start growing in a particular tissue, increasing the amount of the tumor and starts a second step: angiogenesis, formation of new vessels. From them, there’s extravasation, or cells can use lymphatic vessels and start lymphatic metastases. In vitro, tumor cells can be studied:

• Cell proliferation assay (Coulter counter, cell cycle analysis, iCelligence system).
• Cytotoxic assay (MTT, iCelligence).
• Cell migration assay (Boyden’s chamber assay).

Most things seen in tumors can also be seen in atherosclerotic plaques: accumulation of SMC.

Cell proliferation assay

Cells are grown in a tissue culture dish in the presence of medium containing 10% FCS (fetal calf serum). Seeding the cells in 6, 24, or 48 well trays for the experiment:

• All the procedures will be performed under sterile conditions in the biohazard hood.
• Aspirate the medium containing FCS (it stops trypsin) with a sterile Pasteur pipette connected to a vacuum system.
• Wash once with sterile water.