The course

a)Introduction of mammalian cell/tissue culture

b)Monoclonal and polyclonal antibody production and characterization for various applications

c)Adjuvant technology in vaccination and therapy

d)Immunotechnology in cancer therapy and transplantation immunology

e) Stem cell culture and Tissue engineering

Animal Cell Culture

a) Applications of tissue culture

b)

Tissue , organ and cell culture

i)Adult and embryonic tissue, embryonic stem cells ii)Adherent and suspension cell cultures

c)

Stages in cell culture

i) primary cell culture, normal and non-transformed cells, transformed cells

ii) passaging cells iii) Human and rodent cell culture d) Cloning and selecting cell lines

e)

Physical methods of cell separation

What is Tissue culture : In vitro cultivation of organs, tissues and cells is collectively known as tissue culture.
In modern tissue culture techniques standardized media ( DMEM, RPMI, DMEM-F12 etc.) and sophisticated incubation conditions are used.

Using modern cell culture techniques , it is possible to culture different cell types e.g. Connective tissue elements ( fibroblast)

Skeletal tissue ( bone and cartilage)

Cardiac and smooth muscle Epithelial tissue ( liver, lung, breast, skin, bladder, kidney) Endocrine cells ( adrenal, pituitary, pancreatic islets)

Melanocytes and many different type of tumor cells

a) Cells of connective tissues

i) Fibrous connective tissues b) Epithelial cells

cartilage cell ii) Hyaline cartilage connective tissues

Squamous cells(

epithelial

inner lining of

Cubodial epithelial cells

Columner epithelial cells(

intestine

lining

of

small

mouth and blood vessel

(glands and ducts )

c) Muscular Cells

Smooth muscle cells( internal organs and blood vessel )

Striated muscle cells( associated with skeleton )

Applications of cell and tissue culture

a) b) c) Production of monoclonal antibody using hybridoma technology ( Kohler and Milstein 1975) Production of recombinant proteins ( lymphokines, interferons, human growth hormones etc.) and production of viral vectors for gene therapy Reconstitution and replacement of damaged tissue and cells ( vascular tissue, liver assist device for hepatic failure, neural grafts in Parkinsons disease, skin tissue for severe burns, pancreatic islet of Langerhans devices for diabetes) In vitro culture of specific killer T cell, dendritic cell, killer macrophages applicable for therapeutic purposes Cytotoxicity testing( evaluation of anti-cancer drug, effects of carcinogens, toxins, drugs on specific cell types using in vitro models to supplement the animal model) In vitro genetic manipulation of of animals to produce animals with desired characteristics or for cloning desired strains of domestic animals.

d) e) f)

Properties of Animal cells

10-100 microns
Spherical in suspension No cell wall, plasma membrane is thin, fragile and shear sensitive Surface is negatively charged and grown in positively charged surfaces

Downside of Animal cells

Low productivity Good producers 100g/ml/day Average producers 20-100 g/ml/day Poor producers<20 g/ml/day Slow growth rate Low expression rate Complexity of growth conditions Complexity of media

Significance of Mammalian cell Processes

Commercial significance of biopharmaceutical proteins is driving progress in process development
Approximate 40 products made in mammalian cell culture, about half of which are antibody CHO NS0, SP2/0, hybridoma etc BHK C127 - 27 - 8 ( 8 Mab) - 2 - 1

The established cell lines used for pharmaceutical productions :

a) CHO ( Chinese Hamster Ovary cell lines) : These are anchorage dependent cell lines from biopsy of adult Chinese hamster 1957. These cell line has high capacity for amplification and expression of recombinant gene and proteins, ability to synthesize protein with similar glycosylation patterns as observed in native proteins, ability to grow in large scale bioreactors, to facilitate scale up production of transfected cells, CHO cells are pre adapted to serum-free medium Some of the CHO cell line derived products are a) - glutamyl transferase ( diagnostic for hepatic cancer, alcohol abuse), b) IFN-, HIV SP120, c) t-pA, d) Anti thrombin-III, e) Erythropoitin, MCSF At present there are varieties of CHO cell lines e.g. CHO-K1 ( proline requiring mutant), CHOdhFr ( deficient in dihydrofolate reductase)

The established cell lines used for pharmaceutical productions :

b) BHK 21 : This cell line derived from 1 day old Syrian hamster kidney in 1961. These cells are susceptible to virus and extensively used for the production of vaccines ( e.g. foot and Mouth disease Vaccine, FMDV). Some of the products of BHK21 are vaccines, antithrobin III, erythropoitin, Il-2 , transferrin.

c) VERO cells : Derived from Kidney of normal adult African green monkey, susceptible to wide range of viruses( e.g. measles, poliovirus, rubella ) and used for viral vaccine production

d) Namwala / Namwala KJM1 : Human lymphoblastoid cell line, these cells on treatment with Na-butyrate and Sendai virus secretes IFN- and it is well established for industrial manufacturing

Avantages of cell culture in vitro

Single well defined cell type Controlled conditions and easy to manipulate Alternative to animal testing - Model system for discovery of physiological principles - Check latter on the smaller number of animals Large quantity of cells can be obtained

Shortcommings
Not at all like animal - In cell cultures cells are no longer organized into tissues - Monolayer is nothing like a 3D tissue Environment is not like in vivo

- ECM ( extra cellular matrix scaffolding)

Effects of serum plasma etc.
Tissue culture can be a powerful technique if conducted properly and a great waste of time and money when done sloppily Banice Martin

Adult and embryonic tissue, embyonic stem cells

Cultures can be derived from adult tissue or from embryonic tissue.
Cultures derived from embryonic tissue generally survive and grow better than those taken from
adult tissue. Tissues from all parts of embryo are easy to culture, where as tissue from adults are often difficult to culture Widely used embryonic cells are a) mouse embryo fibroblast 3t3 cell lines, b) human fetal lung fibroblast cell line MRC-5

Embryonic stem cells

Embryonic stem cells from embryo during blastocyst stage could be taken out and can be cultured for many generation and are of particular interest because they can be manipulated in culture and then re-introduced into embryos.

Adherent and suspension cell cultures

Adherent cells:
These are anchorage - dependent and attachment to a substratum(with nano scale roughness feature) is prerequisite for proliferation. These are generally subject to contact inhibition. Most cells except mature haemopoietic cells and transformed cells, grow in this way.

Suspension cells :

These anchorage-independent cells and these are originated from blood, spleen, bone marrow. These cells grow in suspension and easier to propagate, since subculture only requires dilution with medium.

Stages in cell culture

primary cell culture, normal and non-transformed cells, transformed cells Primary cell culture : Primary culture a culture started from cells, tissues or organs,
taken directly from organisms. A primary culture may be regarded as such until it is subcultured for the first time and it becomes a secondary culture and subsequently it then becomes a cell line. The cells of the explant or tissue can be isolated and disaggregated by mechanical, chemical, or enzymatic digestion of animal tissue. When these cells are induced to grow in vitro, a primary cell culture results and these cells are generally still representative of the original tissue.

Process for Primary culture:

Disadvantages of Primary cell culture:

a) b) A mixture of cell types is generally present ( since most tissue contain mixtures of cells), in other words it is heterogenous. Primary cell culture requires the recurrent sacrifice of animals

Primary cell culture of rat fibroblast

Enzymes used in enzymatic desegregation

Enzymes - Trypsin, - Collagenase II , elastase ( for fibrous tissue e.g. connective tissue and muscle) - Hyaluronidase ( to dissolve proteoglycans ) - Pronase ( bacterial protease) - DNAse ( to dissolve DNA aggregates from damaged tissue) Usually a combination of enzymes Crude preparations are usually more efficient - The purer the less toxic -The crude the more effective due to contamination with other proteases

Incubation and growth

Appropriate medium supplemented with growth factors, cytokines and other essential metabolites
Some cells require special adhesion surfaces ( cover tissue culture dish with extracellular matrix proteins or synthetic attachment molecules) Transfer cells to final growing conditions as soon as possible

Challenges
- removal of dead cells - enrichment of viable cells - Separation of cell type

Characteristics of normal primary cells

For the safety criteria normal human cell lines are used for vaccine production, because transformed cell lines may transmit carcinogenic agents to the final products. A diploid chromosome, indicating there is no gross genetic change Anchorage dependent and density inhibition, which is an indication of growth control, and is shown by cessation of growth as confluent monolayer on the surface of a growth flask.

A finite life span, which is reflection of intrinsic growth regulation of cells

Non-malignancy of the cells, which can be shown by the inability to form tumors following injection of the cells into immuno-compromised mice

Separation of cell type

Selective media Difference in the speed of attachment Use of enzymes - Collagenase does not easily disperse epithelial cells but work well on stroma Neurons need NGF while glial cells do not

Passaging cells and passage numbers

Adherent cells/ suspended cells :
The kinetics of animal cells growth in vitro follows the similar pattern of bacterial growth kinetics. When the cells are taken( from tissue, primary culture or stationary phase culture)) there is first lag phase of some hours or days before the cells begin to grow. Growth then proceeds steadily with the population doubling every 15-20 hrs in the case of fast growing cells. This phase of exponential growth is known as logarithmic phase. At the end of this phase the maximum population is reached and cells enter the stationary phase or plateau phase in which virtually no growth occurs.
In case of anchorage-dependent cells, cells can be trypsinized and reintroduced into fresh media. This is called passaging or sub culturing. Ideally cells should be passaged during late log phase of cell growth.

Passage number : The passage number is the number of times this procedure is performed
after the original isolation of cells from primary source.

The fate of primary cultures : continuous/immortalized cell line, finite

cell lines, non-transformed cell line, transformed cell line Some
cells are capable of unlimited number of cell divisions in vitro as long as they are supplied with nutrients and there is no limit to the number of passages. These

are described

as continuous or immortalized cell lines.

Some cells ( most deploid cells) are only capable of limited number of cell divisions after which the culture stop dividing and these are called finite cell lines.
Normal cells and non-transformed cell :

The designation of cells as normal implies that growth control is normal, that the cells have undergone no genetic changes, and the cells have not been transformed. Rodent cell lines give rise to continuous cell lines from normal tissue. But these cell line tend to divide faster than cells in vivo, they have slightly reduced cell size, more importantly they are genetically different from original cells, since the culture is *aneuploid. These cell lines are

non-transformed and very useful for

cellular transformation. Treatment of such adherent cells with various agents( viruses, chemicals, radiation) can dramatically change the growth properties of cells in culture and the cell becomes transformed.
*Aneuploidy is a chromosal state in which abnormal numbers of specific chromosomes or chromosome sets exist within the nucleus. Aneuploidy is common in cancerous cells.

Why do primary cells eventually stop dividing in culture?

Telomere shortening
Telomeres are repetitive DNA sequences & associated proteins they caps the ends of chromosomes

they become shorter with each cell division

they are normally maintained by telomerase human fibroblasts do not normally express telomerase they stop dividing when their telomeres get too short

short telomeres send a signal that causes cell cycle arrest

Cell cycle arrest

Cell cycle checkpoint mechanism become activated

Cell cycle stop

Transformed cells
Transformation is defined as the acquisition by cells of certain phenotypic properties associated with cancer. Transformed adherent cells can be identified by changes in the control of their growth behavior.

Phenotypic properties of transformed cells:

Ability to form foci in vitro culture Change in morphology Changes in growth factor requirements Release from anchorage-dependence for cell division Ability to form tumors in vivo

Immortalized cell lines

* Divide indefinitely in culture ( no limit) *Cells are abnormal, but convenient for experiments *Provide unlimited source of genetically identical cells if the cell lines in clonal( arising from a single cell) *cells may be transformed ( made immortal) by introducing oncogene derived from tumor viruses *Oncogene inactivate cell cycle checkpoint mechanisms *Immortal cells can also be isolated from tumors

*normal cells can be transformed by mutagenic chemicals

*Mutations may also inactivate checkpoint mechanisms *Human fibroblasts can be immortalized by artificially expressing telomearse, but this trick does not work with all human cells or with rodent fibroblasts, which normally do express telomerase

Human and rodent cell culture

Hayflick effect ( human cell) : In 1961 , Hayflick and Moorhead studied the potential of
human fetal lung fibroblast growth in vitro. It was observed that after 50 generations the growth of cells begin to slow down and ultimately leads to complete death of culture. The exact number of doubling time depends on the type of cell, its stage of differentiation and origin. The number of population doubling for human cells generally between 20-80 but it can be much shorter. The limited replicative capacity of human cells in culture is sometimes called as Hayflick effect, after its discoverers.

Rodent cell :

Rodent embryo cells routinely give rise to immortal cell lines. When rodent tissues are cultures, there is some initial cell death coupled with emergence of healthy growing cells. As these are diluted and allowed to grow, they soon begin to loose growth potential and most of the cells die. The culture at this stage is said to undergo crisis. However, occasionally a variant population of cells emerges from this phase and these cells continue to grow . These cells constitute an immortalized cell line which will grow forever if appropriately diluted and fed with nutrients. A protocol which is used to establish rodent cell lines is one whereby 3X105 cells are transferred every three days to new dishes. The lines made by this protocol are called 3T3 lines. 3T3 fibroblasts are much used in cancer research.

Human cell line

Rodent cell line

Passage number ( cells are passaged every three days)

Passage number ( cells are passaged every three days)

Phases in the establishment of human and rodent cell culture

Cell Cloning
Primary cel culture is composed of hereogenous cell population of single cell type. A number of methods used to separate different cell type.
The traditional approach is to isolate a pure cell strain from cells in continuous culture by a process called cloning. During cloning, a population of cells is derived from a single cell by mitosis to produce genetically homogenous clone which then can be characterized and stored. Various methods of cloning: Dilution cloning selective media( fibrobl;asts could be eliminated either by
complement-mediated lysis using Mab against fibroblast or by using chemical which suppress fibroblast overgrowth e.h. Cis-OH proline, Na-ethyl mercuri thio salicylate, phenobarbitone)

cell separation by FACS