With thanks to all those who viewed and appreciated my "Animal Cell Culture : In a nut shell" presentation.....

2. Cell culture is the complex process by which cells are grown under controlled
conditions. In practice, the term "cell culture" has come to refer to the culturing of cells
derived from multicellular eukaryotes, especially animal cells
Animal cell culture is playing a vital role in the various modern researches.
Recent developments in the field of biotechnology and molecular biology have given
various new dimensions to this technology.
Cell culture has provided a new tool for the better understanding of diagnosis and
molecular biology in many areas of biological systems including bacterial, viral and
parasitic diseases.

4. o The primary impetus for the development of cell culture was to
study, under the microscope, normal physiological events of cells.
o Haberlandt (1902) stated that the in vitro-culture techniques for
plants primarily to facilitate basic physiological research.
o Ross Granville Harrison (1907) developed a culture to study the
development of nerve fibers using Frog as a source.
o Alexis Carriel (1912) used tissue and embryo extracts as culture
media to keep the fragments of chick embryo heart alive.
o Mammalian cell cultures can be a suitable alternative for the use of
whole animal tests to establish the potential toxicity of compounds.
HeLa (Henrietta Lacks) cells
one of the earliest human cell lines cultured
by George Otto Gey to create
an immortal cell line for medical research.

5. o This is due to many reasons:
1. They can overcome the disadvantages of the whole animal tests viz.;
▫ High costs. ▫ Variability of results.
2. Growing moral/ethical objections for the use of animals in toxicity
testing.
3. Cell culture tests are rapid, allow more efficient screening of novel
compounds and sometimes can allow the identification of metabolic
targets of inhibition.
o Cell culture tests can be designed to evaluate various effects:
▫ Reduced growth rate.
▫ Breakdown of membrane permeability
▫ Tissue specificity of response.
▫ Ability to metabolize toxic compounds.
▫ Genetic effects/mutagenicity.

7. • Water Jacketed CO2 incubator
• Gas/CO2 Incubator with RH Control
Precise control of Oxygen levels combined with
CO2, N2 and RH ensure accurate conditions for
applications such as, hypoxic cell studies and
cancer research.

9. • HEPA filter rated at 99.99%
efficient for 0.3 micron
particulates. The HEPA filtered
air is then directed vertically
across the work surface.
Filter sterilization
Media that cannot be autoclaved
must be sterilized through a 0.22 mm
pore size membrane filter.

10. o Dishes
o Multi-well plates
o Flasks (vented &
non-vented capped)
o Flasks on slide
 Filtered vents on caps
(aids entry of CO2)
 Hydrophilic bottom
(helps attachment of cells)

13. • A growth medium or culture medium is a liquid or gel designed to
support the growth of microorganisms or cells. The components
of a suitable culture media include:
• Basic media
• Buffering capacity
• Glutamine & other amino acids
• Serum
• Antibiotics & Anti-mycotics
• The choice of culture media used will depend on the type of primary
cell, cell line, and the incubation conditions.
• Culture media have a limited storage life and the recommendations
indicated by the supplier should be followed.
• Culture media can be supplied in powdered form which requires
dissolving and filter sterilizing.
• Bottles of media should be prepared in small batches, for instance
two weeks supply at a time.

14. • Maintain pH and osmolarity (260-320mOsm/L).
• Provide nutrients and energy source.
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
Contains a pH indicator (Phenol red) Media looks
pink/red at pH 7.2
Acidic -yellow /orange (cell growth, bacterial
growth)
Basic -purple (no cell growth, not enough CO2)
Buffers (Bicarbonate and HEPES)
• Bicarbonate buffered media requires CO2
atmosphere
• HEPES Strong chemical buffer range pH 7.2 - 7.6
(does not require CO2)
Glucose
• Energy Source
• Growth factors and hormones
• Aids cell attachment
• Binds and neutralise toxins
• Long history of use
• Infectious agents (prions)
• Variable composition
• Expensive
• Regulatory issues (to minimise risk)
Destruction of complement and immunoglobulins
Destruction of some viruses
(also gamma irradiated serum)

15. Keto acids (oxalacetate and pyruvate)
• Intermediate in Glycolysis/Krebs cycle
• Keto acids added to the media as additional
energy source
• Maintain maximum cell metabolism
Carbohydrates
• Energy source
• Glucose and galactose
• Low (1 g/L) and high (4.5 g/L) concentrations
of sugars in basal media
Vitamins
• Precursors for numerous co-factors
• B group vitamins necessary for cell growth
and proliferation
• Common vitamins found in basal media is
riboflavin, thiamine and biotin
Trace Elements
• Zinc, copper, selenium and tri-carboxylic acid
intermediates
L-glutamine
• Essential amino acid (not synthesised 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)
• Usually added to basic media
compositions
• Energy source, used in protein synthesis
• May reduce metabolic burden on cells
Growth Factors and Hormones (e.g.: insulin)
• Stimulate glucose transport and utilisation
• Uptake of amino acids
• Maintenance of differentiation
Antibiotics and Antimycotics
• 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

16. • Primary culture ; a cell or tissue culture started from material taken directly
from an organism.
• Secondary culture ; a cell or tissue culture started from material taken directly
from a Primary culture.
• Cell line: A cell line is a permanently established cell culture that will proliferate
indefinitely given appropriate fresh medium and space
Different cell types which can be grown in culture include;
• Connective tissue elements,
• Skeletal tissue (bone and cartilage),
• Cardiac and smooth muscle,
• Epithelial tissue (liver, lung, breast, skin, bladder and kidney)
• Endocrine cells (adrenal, pituitary, pancreatic islet cells)
• Tumor cells.
Cells may grow as an adherent monolayer or in suspension. Adherent cells are
said to be anchorage-dependent and attachment to a substratum is a prerequisite
for proliferation. They are generally subjected to contact inhibition, which means
they grow as an adherent monolayer and stop dividing when they reach such a
density that they touch each other.

17. Check confluency of cells
Remove spent medium
Wash with PBS
Resuspend in serum
containing media & Transfer to
culture flask
Incubate with Trypsin/EDTA
(TRED)
70-80% confluence 100% confluence
Resected Tissue
Cell / Tissue culture in vitro
Primary culture
Secondary culture
Sub-culture
Cell Line
Sub-culture
Immortalization
Successive sub-culture
Single cell
Isolation
Clonal cell line
Senescence
Transformed
cell line
Immortalised cell line
Loss of control
of cell growth

19. • Affected cultures should be disposed into 2.5% hyper-chlorite
solution.
• Media bottles known, or suspected, to be contaminated should
be disposed of as well.
Some basic preventive measures may be taken:
1. checking sterilizing procedures (autoclave and oven procedures)
2. checking sterility of laminar flow hoods,
3. Regular checks on cultures;
4. disposal of contaminated cultures rather than attempting to
decontaminate them.
Passage cells
Centrifuge &
Aspirate supernatant
Transfer to cryo-vial
Freeze at -80oC
Resuspend cells in
10% DMSO in FCS
Transfer to liquid
nitrogen storage tank
Resuspend cells in serum
containing media
Chemical Contamination
Media
Incubator
Serum
water
Biological Contamination
Bacteria and yeast
Viruses
Mycoplasmas
Cross-contamination by other cell culture

20. Cell morphologies vary depending on cell
type
Fibroblastic
Endothelial
Epithelial
Neuronal
Cardiac muscle

21.  A primary culture is that stage of the culture after isolation of the cells
but before the first subculture.
 There are four stages to consider:
1) Acquisition of the sample
2) Isolation of the tissue
3) Dissection and/or disaggregation
4) Culture after seeding into the culture vessel
 A primary cell culture may be obtained either by allowing cells to
migrate out from fragments of tissue adhering to a suitable substrate
or by disaggregating the tissue mechanically or enzymatically or by
treatment with chelating agents to produce a suspension of cells.
CellNumber
Passage Number Age (years)
Passes

22. • Collagenase - the extracellular matrix often contains collagen, particularly in connective tissue and muscle.
• Hyaluronidase – to dissolve proteoglycans
• Pronase and Dispase – bacterial proteases
• DNase – to dissolve DNA aggregates from damaged cells

23.  Physical disruption, which may involve cutting the tissue into pieces, is often
combined with other methods, but chick embryo suspension may also be obtained
by expressing (squeezing) the embryo through the nozzle of a syringe.
 Tissues like epithelium (which needs Ca++ and Mg++ ions for its integrity) can be
treated with chelating agents, such as Citrate and Ethylene di-amine tetra acetic acid
(EDTA). Chelating agents are mainly used for production of cell suspensions from
established cultures of epithelial type.
 The enzymes used most frequently for tissue disaggregation are crude preparations
of trypsin, collagenase, Elastase, Pronase, Dispase, DNase, and Hyaluronidase, alone
or in various combinations. e.g., Elastase and DNase for type II alveolar cell isolation.
 There are other, non-mammalian enzymes, such as Trypzean (Sigma), a recombinant,
maize-derived, trypsin also available for primary disaggregation.
 Trypsin and Pronase give most complete disaggregation, but may damage the cells.
 Collagenase and Dispase, on the other hand, give incomplete disaggregation, but are
less harmful.

24. o Fat and necrotic tissues are best removed during dissection.
o The tissue should be chopped finely with sharp instruments to cause
minimum damage.
o Enzymes used for disaggregation should be removed subsequently
by gentle centrifugation.
o The concentration of cells in the primary culture should be much
higher than that normally used for subculture
o A rich medium, such as Ham’s F12, is preferable to a simple medium.
o Embryonic tissue disaggregates more readily, yields more viable
cells, and proliferates more rapidly in primary culture than does
adult tissue.
• Make sure that your work fits within medical ethical rules or current
legislation on experimentation with animals.
• Work with human tissue should be carried out at Containment Level
2 in a Class II biological safety cabinet.
• sterilize the site of the resection with 70% alcohol if the site is likely
to be contaminated (e.g., skin).
• Remove the tissue aseptically and transfer it to the tissue culture
laboratory in dissection BSS (DBSS) or transport medium.
• Do not dissect animals in the cell culture laboratory, as the animals
may carry microbial contamination.
• The tissue can be hold held at 40C for up to 72 h, although a better
yield will usually result from a quicker transfer.

25.  Mouse embryos are a convenient source of cells for undifferentiated
fibroblastic cultures. They are often used as feeder layers.
Outline: Remove uterus aseptically from a timed pregnant mouse and dissect
out embryos.
Materials
 Timed pregnant mice
 DBSS
 BSS
 Petri dishes
 Pointed forceps
 Pointed scissor
 70% alcohol
 Laminar air flow
 Bunsen burner

26. PROTOCOL
1.Male & Female mice are put together for mating, estruses are induced in female 3days later.
The time of successful mating is determined by examining the vagina for a hard, mucous plug.
2.The day of detection of a vaginal plug, or the ‘‘plug date,’’ is noted as day zero, and the
development of the embryos is timed from this date.
• The optimal age for preparing cultures from whole disaggregated embryo is around 12 days.
3. Kill the mouse by cervical dislocation, and swab the ventral surface liberally with 70% alcohol.
4.Tear the ventral skin transversely at the median line just over the diaphragm, grasping the skin
on both sides of the tear, pull in opposite directions to expose untouched ventral surface of
the abdominal wall.
5.Cut longitudinally along the median line of the exposed abdomen with sterile scissors,
revealing the viscera. At this stage, the uterus, is obvious in the posterior abdominal cavity.
6. Dissect out the uteri into a 25ml or 50ml screw-capped vial containing 10-20ml DBSS.
• All of the preceding steps should be done outside the tissue culture laboratory; a small laminar-flow
hood and rapid technique will help to maintain sterility.
7.Take the intact uteri to tissue culture lab & transfer them to a fresh Petri dish of sterile DBSS.
8.Dissect out the embryos:
(a) Tear the uterus with two pairs of sterile forceps, keeping the points of the forceps close together to
avoid distorting the uterus and bringing too much pressure on the embryos.
(b) Free embryos from the membranes and placenta & place them on side of a dish to bleed.
9.Transfer the embryos to fresh Petri dish kept on ice (for subsequent dissection and culture).

28. • Chick embryos are easier to dissect, as they are larger than mouse embryos
at the equivalent stage of development
• chick embryos are used to provide predominantly mesenchymal cell primary
cultures for cell proliferation analysis, to provide feeder layers, and as a
substrate for viral propagation.
• it is easier to dissect out individual organs to generate specific cell types, such
as hepatocytes, cardiac muscle, and lung epithelium
Outline: Remove embryo aseptically from the egg and transfer to dish.
Materials
• DBSS
• Forceps, straight and curved
• Small beaker
• Petri dishes
• Embryonated eggs
• Alcohol
• Humid incubator

29. PROTOCOL
• Incubate the eggs at 38.50C in a humid atmosphere, and turn the eggs through 180 degree
daily.
• Swab the egg with 70% alcohol, and place it with its blunt end facing up in a small beaker
• Crack the top of the shell, and peel the shell off to the edge of the air sac with sterile
forceps.
• Re-sterilize the forceps and then use the forceps to peel off the white shell membrane to
reveal the chorio-allantoic membrane (CAM) below, with its blood vessels.
• Pierce the CAM with sterile curved forceps, and lift out the embryo by grasping it gently
under the head. Do not close the forceps completely, or else the neck will sever, use the
finger pad to restrict the pressure of the forefinger.
• Transfer the embryo to a 9-cm Petri dish containing 20 mL DBSS and preserve it for
preparation of culture.

31. 488 nm laser
+-
FALS Sensor
Fluorescence detector

32. Magnetic separationMagnetic labeling Elution of the labeled
cells (positive fraction)
Isolation of untouched cellsMagnetic labeling of non-target cells

33. Cancer is an umbrella term covering a plethora of conditions characterized by
unscheduled and uncontrolled cellular proliferation.
• Carcinomas; constitute 90% of cancers, are cancers of epithelial cells
• Sarcomas; are rare and consist of tumors of connective tissues
(connective tissue, muscle, bone etc.)
• Leukemias and lymphomas; constitute 8% of tumors. Sometimes
referred to as liquid tumors. Leukemias arise from blood forming cells
and lymphomas arise from cells of the immune system (T and B cells).
• Ionising radiation – X Rays, UV light
• Chemicals – tar from cigarettes
• Virus infection – papilloma virus can be responsible for cervical cancer.
• Hereditary predisposition – Some families are more susceptible to getting
certain cancers. Remember you can’t inherit cancer its just that you maybe
more susceptible to getting it.

34.  Nomenclature describing several cell lines has not yet been standardized.
 The problems are both linguistic and experimental. Many ambiguous cell line names appear
in the published literature.
 Cell line knowledgebase (CLKB) with a well-structured collection of names and descriptive
data for cell lines cultured in vitro, is a step towards rationalizing the nomenclature.
 CLKB contains broad collection of cell-line names compiled from ATCC, Hyper-CLDB & MeSH.
 In addition to names, the knowledgebase specifies relationships between cell lines.
 The CLKB facilitates data exploration and comparison of different cell lines in support of
clinical and experimental research.

35. Human cell lines
• MCF-7 Breast cancer
• HL 60 Leukemia
• HEK-293 Human embryonic kidney
• HeLa Henrietta lacks
Primate cell lines
• Vero African green monkey
kidney epithelial cells
• Cos-7 African green monkey
kidney cells
And others such as CHO from hamster, sf9 & sf21
from insect cells

36. • Calcium phosphate precipitation
• DEAE-dextran (dimethylaminoethyl-dextran)
• Lipid mediated Lipofection
• Electroporation
• Retroviral Infection
• Microinjection

37. o Primary cells only undergo a pre-determined and finite number of cell divisions in culture
and enter a state where they can no longer divide (Replicative senescence).
o Replicative senescence is marked by distinct changes in cell morphology, gene expression,
and metabolism like Increased cell size , development of multiple nuclei , activation of
tumor suppressor proteins like p53, RB, and p16 , increased lysosomal biogenesis and
over-expression of endogenous β-galactosidase.
o In order to have consistent material throughout a research project, researchers need
primary cells with an extended replicative capacity, or immortalized cells with similar or
identical genotype and phenotype to their parental tissue.
Several methods exist for immortalizing mammalian cells in culture conditions.
 One method is to use viral genes, such as the Simian Virus 40 (SV40) T antigen, to induce
immortalization. Recent studies have also shown that SV40 T antigen can induce
telomerase activity in the infected cells.
 The most recently discovered approach to cell immortalization is through the expression
of telomerase reverse transcriptase protein (TERT)
 This protein is inactive in most somatic cells, but when hTERT is exogenously expressed,
the cells are able to maintain sufficient telomere lengths to avoid replicative senescence.
 Recently scientists have developed a comprehensive cell immortalization technique that is
comprised of ready-to-use recombinant Retroviral, Lentiviral and Adenoviral vectors for
hTERT, p53 and RB and SV40 T antigens to make cell immortalization easier. This technique
confirms the successful immortalization by virtue of antibiotic selection markers.

38. • While many cells make up simple body parts, such as tissues, some complete
more complex and specialized tasks.
•Each of these cell types are formed and operate differently, ensuring that the
cell can carry out the necessary body function that it is intended to complete.
• Disaggregation : Gentle mechanical disaggregation or collagenase digestion in
preference to trypsinization. Collagenase, in particular, appears to give good survival as it
does not completely dissociate the epithelium but frees it from the surrounding stroma.
• Selective Detachment: Selective detachment has also been employed, and enzymes
such as dispase can release epithelial sheets before the fibroblasts. EDTA can also be used
to remove fibroblasts selectively from mixed cultures of keratinocytes
• Substrate Modification: Collagen coating, laminin coating, Matrigel , Becton Dickinson
produces a modified plastic, Primaria, that has a net positive charge claimed to favor
epithelial growth in preference to fibroblasts.
• Feeder Layers : The most popular substrate modification is to preplate with a monolayer
of fibroblasts, or other cells, that can be irradiated or mitomycin C-treated to prevent
their further growth.
• Cell Separation: It is possible to separate many cell types by physical methods, such as
density gradient centrifugation, centrifugal elutriation, and flow cytometry. Magnetic
separation with Specific antibodies conjugated to iron-containing coated beads.
• Selective Culture: Selective media with growth factors.

39. Effectors
Complement (C’)
Macrophages (Mf)
Granulocyte
Natural Killer (NK) cells
Cytotoxic T lymphocytes (CTL)
Target Cells
Tumors
Virally infected cells
Cells infected with intracellular
bacteria
Monocyte - blood
Alveolar Mfs–lung
Histiocytes – connective tissue
Kupffer cells – liver
Mesangial cells – kidney
Microglial cells – brain
Phagocytosis
Antigen Processing and
Presentation
Cytotoxicity
Direct Cytotoxicity
Antibody dependent cell-
mediated cytotoxicity (ADCC)
Reactive oxygen intermediates (ROIs): O2
-, OH-, H2O2
Reactive nitrogen intermediates (RNIs): NO, NO2
Tumor necrosis factor-a
Lysosomal enzymes
Target
Fc receptor
Mf
Ag
Ab

40. Beneficial and Deleterious Effects of
Cytotoxicity
Protection against
– Tumors
– Virus-infected cells
– Intracellular bacteria
– Parasites
– Fungal infections
Apoptosis
(Programmed cell death)
 Nuclear and cytoplasmic
condensation
 Membrane blebs
 DNA fragmentation
(early, 180bp multiples)
 Apoptotic bodies
 Phagocytosis
 Localizes infection
Ex. Development
3H-thymidine-release assay
Necrosis
(Pathologic cell death)
 Cell Swelling and lysis
 Inflammation
 DNA fragmentation (late,
varying size)
 Spreads infection
Ex. Stress
51Cr-release assay
Role of Apoptosis
• Embryogenesis
• Organogenesis
• Cytotoxic Lymphocyte killing of targets
Fas
FasL
Ag
MHC
TCR
Perforin
Granzyme
CTL
Cause
Autoimmune disorders
Transplant rejection
Immunopathology

41. • Cell viability is determined by staining the cells with Trypan blue
• As Trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable
to this dye
• Stain the cells with Trypan dye and load to Haemocytometer and calculate % of viable cells
% of viable cells = No. of unstained cells x 100
total no. of cells

43. Fusion of two different cells to produce a hybrid cell.
 Macrophages fuse around the foreign body or bacterial cell in the tissues.
Phagocytosis
 Bones cells also known to undergo somatic fusion. Osteoclasts
 In culture cells are induce to fuse by some viruses e.g. Sendai virus
This virus induces first to form heterokaryon and during mitosis chromosomes of heterokaryon
are brought towards poles and which latter on to form hybrid.
 Some chemicals such as polyethylene glycol also induce somatic cell fusion.
Hybridoma technique
 Taxonomically different animals can fuse and form hybrids. SCH
Application in biotechnology:
• Study and control gene expression and differentiation.
• Gene mapping
• Malignancy
• Viral replication
• Antibodies production via hybridoma technology

44. A) Production of vaccines:
Two factors stimulated the use of tissue cultures for vaccine production:
▫ The ability to grow viruses in cell cultures.
▫ Current egg-vaccine production requires long time (9 months) that hinder
the response to unanticipated demands.
o In (1949), Enders discovered that the poliomyelitis virus could be grown
from primary monkey cells in culture.
o The polio vaccine, produced in 1954, was the first human vaccine to be
produced using large-scale cell culture techniques.
o Animal cell technology is
considerably developed for
the production of a range of
human and veterinary viral
vaccines against a variety of
diseases.

45. B) Production of antibodies:
o Also, the in vitro methods for production of mABs are the methods of choice
because of:
▫ The ease of culture for production.
▫ Less economic consideration compared with the use of animals.
o These advantages make the in vitro methods meet more than 90% of the
needs for mABs.
o The ability to generate hybridomas has stimulated the use of the in vitro
methods for mABs production (B lymphocyte-myeloma cell hybrid).
 Practical uses of the in vitro produced mABs:
▫ Diagnostic tests for the identification of small quantities of specific
antigens.
▫ mABs also are used therapeutically: OKT3 recognizes a surface antigen
(CD3) on T cell and is one of the most effective agents in preventing
immunological rejection of transplanted kidneys.

46. ▫ Various mAbs designed to destruct tumor cells by targeting a membrane
bound protein antigens specifically expressed by these cells.
▫ The conjugation of radioactive or toxic compounds to the antibody can result
in a localized cytotoxicity to the target cells.

48. YEAR Product Target Indication
1986 Orthoclone CD3 Transplant Rejection
1994 ReoPro GPIIa/IIIb Angioplasty
1997 Rituxan CD20 B Cell Lymphoma
1998 Zenapax IL2R Transplant Rejection
1998 Simulect IL2R Transplant Rejection
1998 Remicade TNF Crohn’s, RA
1998 Herceptin Her2 Breast Cancer
2000 Mylotarg CD33 AML
2001 Campath CD52 CLL
2002 Zevalin CD20 B Cell Lymphoma
2003 Bexxar CD20 B Cell Lymphoma
2003 Raptiva CD11a Psoriasis
2004 Avastin VEGF Colon Cancer
2004 Erbitux EGFR Colon Cancer

49.  Examples for biologically important products:
1- Interferon:
Discovered when Isaacs and Lindenmann (1957) found that culture medium
taken from cells that had supported viral growth could protect non-infected
cells from a subsequent viral infection.
2- Tissue plasminogen activator (t-PA):
t-PA was produced in large scale by Genenteck from transfected CHO-K1 cells. It
is used to prevent undesirable formation of fibrin clots in the bloodstream.
3- Blood clotting factors:
For example, factor VIII is produced in large scale by Bayers through
transfection of the mammalian kidney cell line (BHK) with an appropriate gene.
C) Recombinant proteins:
o This idea based on the ability to transfect cells with isolated genes
and amplify it to allow high level of expression of the corresponding
proteins.
o Proteins extracted from biological sources have been important for
the substitution therapy since the 1920s when Best and Banting
used insulin to treat diabetes.

51. Saints Cosmas and Damian
performing a miraculous
transplantation
Oil painting on panel 168 x 133 cm.,
attributed to the Master of Los
Balbases, Burgos, Spain, c. 1495

52. Tissue engineering means the re-constitution of human tissues from
the combinations of cell types grown in culture. This is an important prospect for
future therapeutic treatment with organ failure.
Artificial tissues:
o The re-constitution of skin formed from two layers derived from cultured
human cells:
▫ A dermal-equivalent formed from fibroblasts.
▫ An epidermal-equivalent which is layered on the dermal surface.
o Bio-artificial bone tissues made of Osteoproginater
Tissue
engineered Skin
Tissue engineered bone
•Wound visualization
•Quick and easy application
•Reduced time to wound healing
•Cost savings when compared with comparable
treatments
•Silicone barrier decreases the risk of infection

53. Blood contacting
device
Blood contacting material No. implanted
per year
Vascular graft Dacron (PET), Gortex (PFTF) 200,000
Stents Stainless steel 4,000,000
Catheters Silicone, polyurethane, Teflon 200,000,000
Heart valve cuff Dacron (PET) 200,000
Tubing Silicone
Blood-Contacting Devices
Arteriovenous (AV) GraftsVascular Grafts
Stents

54. Artificial organs:
Construction of organs in in vitro have met technical difficulties:
▫ Multiple cell types require complex scaffolds and an extracellular matrix to support
the functional relationship between cells.
▫ Multiple cell layer require a nutrient supply equivalent to blood capillaries in vivo.
The Vacanti mouse, which came from the Vacanti-
Langer collaboration was one of the first examples of
tissue engineering to catch public attention, and it
probably did more to encourage the early funding for
research in regenerative medicine.
Bio-artificial bladder made of tissue sample surgically
removed from the patient, supported by a biodegradable
scaffold made of collagen.
Developed by Antony Atala and his team at Wake Forest
University
University of Minnesota in 2007 – heart perfusion;
using stem cells created a “new heart”.

55. • Regenerative Medicine is the scientific field that focuses on new approaches to the
autologous repair and/or replacement of cells, tissues and/or organs.
• Broad research area with several main focuses:
– Cellular Therapies
– Gene Therapies
– Tissue Engineering
Cellular Therapies
• Literally, cell therapy means treatment with cells, i.e. replacing diseased or dysfunctional
cells with healthy functioning ones.
• Cellular therapies have the promise to become major therapeutic modalities of the next
century.
• However, cellular therapy is not a new concept:
– blood transfusions routinely performed for several decades
– RBC’s to anemic patients to restore O2 transport
• Examples:
– Bone marrow transplantation (currently performed)
• When hematopoietic cells are vulnerable to destruction by cytotoxic drugs (chemotherapy) to
eradicate residual tumor cells. Bone marrow pluripotent stem cells can be isolated and
expanded prior to chemotherapy to provide a source of mature hematopoietic cells.
– Chondrocyte transplantation (in clinical trials)
– Pancreatic b-islet transplantation (in clinical trials)

56. Gene Therapy
• Gene Therapy is the technique for correcting defective genes responsible for disease
development.
• Genes
– carried on chromosomes; the basic physical and functional units of heredity
– specific sequences of bases that encode how to make proteins
– when altered, encoded proteins are unable to carry out their normal functions, genetic disorders
can result
• Several approaches are currently under investigation:
– insertion of the gene into a non-specific location within the genome to replace a non-
functional gene (most common)
– homologous recombination to swap abnormal gene with a normal gene
– selective reverse mutation to return the abnormal gene to its normal function
– alteration of gene regulation (degree to which a gene is turned on or off)
• In most gene therapy studies, a "normal" gene is inserted into the genome in vivo or in
vitro to replace an "abnormal," disease-causing gene .
• A carrier molecule (vector) must be used to deliver the therapeutic gene to the patient's
target cells. Currently, the most common vectors used are viruses which have been
genetically altered to carry normal human DNA.
• Viruses have evolved a way of encapsulating and delivering their genes to human cells
(pathogenic) and scientists have tried to harness this capability and manipulate the viral
genome to deliver therapeutic genes.

57. In late 1998, James Thompson
at UW-Madison discovered how
to isolate and culture hES cells.

59.  Undifferentiated Cells
 Found throughout the body after embryonic
development
 Multiply by cell division to replenish dying cells
 Regenerate Damaged Tissues.
o Hematopoietic
o Mammary
o Mesenchymal
o Neural
o Endothelial
o Olfactory
o Neural crest
o Testicular
• Identified in many organs and tissues -
Brain, Bone marrow , Peripheral blood, Blood vessels, Skeletal
muscle, Skin, Teeth, Heart, Gut, Liver, Ovarian epithelium and Testis
• Thought to reside in a specific area of each tissue - a ‘stem cell niche’
Leukemia
• bone marrow transplants
Parkinson's Disease
• disorder of the CNS
• lack a sufficient amount of dopamine
• multiply cells that release dopamine
Cardiologists worldwide
currently use VesCell™ adult
stem cell therapy to treat
patients suffering from coronary
artery disease, cardiomyopathy
and congestive heart failure.
Thalesemia
• The genetic defect results in reduced
rate of synthesis of one of the globin
chains that make up hemoglobin
• Hematopoietic stem cell transplantation
(HSCT) is the only curative approach

63. Nuclear Transfer

64. nucleus
skin cells
egg
embryonic stem cells
differentiated donor cells
early embryo
?
“Therapeutic” cloning
transplantation

65. nucleus
adult cells
enucleated oocyte
iPS cells
differentiated donor cells
transplantation
blastocyst
Induced pluripotent stem (iPS) cells
Oct4
Sox2Klf4
c-Myc

66. Limitations of adult stem cell
• The isolation of some types of ASC, for example the isolation of neural cells
from a patient's brain, would be impractical
• Where a person suffers from a genetic disorder or some types of cancers, ASC
isolated from that individual will retain the damaging genetic alterations
underlying the disease and so be of little therapeutic value
• Unambiguous identification is difficult
• Maintenance in culture is difficult
Benefits of Adult Stem Cell Research
• Easy to obtain
• Potentially limitless in supply
• Patients can use their own stem cells for treatment and therapy
• Adult stem cells are politically neutral
• Not offensive to any major interest group nor do they generate controversy.

68. Do clones occur naturally?
• Bacteria, protozoa, yeast produce genetically
identical offspring by asexual reproduction.
• Clones in Plants are formed asexually by
vegetative propagation & apomixis
• In higher animals, clones occur naturally by
parthenogenesis
• Formation of identical twins due to the
splitting of fertilized egg can be called as
clones in higher mammals and humans
• Term clone is derived from Greek word - κλῶνος (Pr: klonosh) “trunk/Branch”
• The term cloning describes a number of different processes (either natural or
artificial) that can be used to produce genetically identical copies of a biological
entity. The copied material, which has the same genetic makeup as the original, is
referred to as a clone.
•Gene cloning : produces copies of genes or segments of DNA
•Reproductive cloning : produces copies of whole animals
•Therapeutic cloning : produces embryonic stem cells for experiments aimed at creating tissues

69. Embryo splitting / twinning :
This involves dividing an eight cell embryo into single cells or blastomeres. Transferring
two of these blastomeres into an empty ZONA PELLUCIDA creates an embryo.
Hans Dreisch - Sea urchin, Hans Spemman - Salamander

70. Somatic Cell Nuclear Transfer (SCNT) Method
This involves transferring the nucleus of an adult somatic cell into an
enucleated egg cell. The cell is then cultured until it divides a number of times to
form a blastocyst or early embryo.
• First explored by Hans Spemann in the 1920's
• The egg cell is enucleated, eliminating the majority of its genetic information
• The donor cell is then forced into the Gap Zero, or G0 cell stage
• The (2n) donor nucleus is then placed inside the enucleated egg cell, either by cell
fusion or transplantation

71. Roslin method:
• It is a variation of SCNT that was developed by Ian Wilmut and Keith Cambell at the
Roslin Institute. The researchers used this method to create Dolly
• somatic cells (with nuclei intact) are allowed to grow and divide and are then deprived of
nutrients to induce the cells into a suspended or dormant stage
• synchronize the cell cycles of the donor cell and the egg cell
• The enucleated egg is placed in close proximity to a somatic cell and both cells are
shocked with an electrical pulse. The cells fuse and the egg is allow to develop into an
embryo which is later implanted into a surrogate (Success rate : 1: 277)

72. The Honolulu Technique :
• developed by Dr. Teruhiko Wakayama et al at the
University of Hawaii
• Produced 3 generations of genetically identical cloned mice
• Sertoli cells, brain cells, and Cumulus cells were used
• The donor nuclei were isolated within minutes of the each
cell’s extraction from a mouse.
• No in vitro, or outside of an animal, culturing was done on
the cells
• The donor nuclei was inserted into the enucleated egg cells
• After one hour, the cells had accepted the new nucleus
• After an additional five hours, the egg cell was then placed
in a chemical culture to jumpstart the cell’s growth also
containing cytochalasin B
• After being jumpstarted, the cells develop into Embryos
• Then transplanted into surrogate mother and carried to term
• Wakayama also made clones of clones and allowed the
original clones to give birth normally (Success rate : 3 : 100)

73. • Upgrading herd quality in farmed animals
• Endangered species can be preserved
• Human therapeutic proteins can be
produced
• Animal models of human disease
• Basic research on cell differentiation
• Disease resistance of farm animals
• Therapeutic cloning for humans
• Assisted reproduction

74. • Inappropriate donor or recipient cell
• Absence of synchrony between the 2 cell’s cycle phases
• Damage from handling
• Not proper reprogramming
From 277cell fusion, 29 early embryo develop & implant in 13 surrogate mother,
only 1 pregnancy went to full term & Dolly born.
• cloning expensive and highly inefficient
• > 90% of cloning attempts fail to produce
viable offspring
• High rate of fetal loss during pregnancy
• Higher rates of infection
• Early neonatal death
• Suffering with lots of syndrome.
• Tumor growth
 Cloning of pets (cats, dogs, horses)
 for Production of transgenic animals
 Cloning humane disease study
 For production of pharmaceuticals like Factor
IX protein in milk, issue plasminogen activator
 Xenotransplantation:-to develop an animal
whose organ will not cause immunological
response

75. • Infertility is a very common condition affecting approximately 13-14% of
couples in the reproductive age group.
• Although this prevalence has remained stable over the last few years, the
demand for infertility services has increased substantially.
• This increase is due primarily to the Baby Boom generation entering into the
reproductive age group at a time of highly publicized technological advances.
Definition of Infertility & ART
“Infertility is defined classically as the inability to conceive after 1 year of
unprotected intercourse. This definition is based on the cumulative probability of
pregnancy”
Who is eligible for ART
• Women with tubal diseases
• Unexplained infertility
• Endometriosis
• Immunologic causes for infertility
• Women with premature ovarian failure
• Individuals with male factor infertility (e.g., abnormalities in sperm
production, function or transport or prior vasectomy)

76. 1.Investigation of possible causes: Before any treatment, women undergo tests for their levels of
FSH (follicle stimulating hormone) and LH (luteinising hormone); men have their semen analysed.
2. Boosting egg supply: At the start of treatment, drugs block the hormones the pituitary gland
usually produces during a monthly cycle. This allows better control over when eggs are produced.
Stimulating drugs are then taken to make the ovaries produce more than one egg.
3. Checking on development: Vaginal ultrasound scans are carried out to monitor developing
follicles.
As soon as tests show these are sufficiently large, another injection of a different hormone helps the
eggs mature. Timing is crucial, as this injection must be given 34-38 hours before eggs are collected.
4. Egg collection: Eggs are collected by ultrasound guidance or, occasionally, by laparoscopy.
A thin needle is inserted through the vagina into each ovary, then into each follicle in turn, sucking
any mature egg into it.
5.Sperm collection: Around the time eggs are collected, the man produces a fresh sample of sperm.
This will be stored for a short time before the sperm are washed and spun at a high speed so that
the healthiest and most active can be selected.
6. Fertilisation: The eggs and sperm are mixed and left in a laboratory dish for 16-20 hours before
they are checked to see if any have fertilised. Any that haven’t, or any that have fertilised
abnormally, are discarded.
The remaining embryos are then left for another 24-48 hours before being checked again.
7. Preparing for pregnancy: Progesterone pessaries help prepare the lining of the womb.
8. Embryo transfer: Two to five days after fertilisation, one or two healthy embryos (three if the
woman is over 40) are usually chosen and put back into the womb via
a catheter. Steroids may be prescribed to help with implantation.
Remaining embryos may be frozen for future IVF attempts, if they are suitable

77. • ZIFT
Zygote Intrafallopian Transfer. ZIFT may be recommended if the husband has severe male fertility factor or if
there has been difficulty confirming fertilization with past procedures. ZIFT has the advantages of allowing
fertilization to be confirmed and it has demonstrated higher success rates than IVF when used for the
appropriate indications.
One disadvantage with ZIFT is that the transfer of the zygote must be performed through a laparoscope.
• GIFT
Gamete intrafallopian transfer was developing in 1984 as a variation of in vitro fertilization (IVF).
Gift is recommended when
• unexplained infertility
• infertility due to immunological factors
• endometriosis
• selected cases of male infertility
• Tubal infertility
A requirement for the procedure is that the female partner having at least one open (patent) fallopian tube.
• TEST
Tubal Embryo Transfer; the placing of cleaving embryos into the fallopian tube.
• POST
Peritoneal Oocyte and Sperm Transfer; the placement of oocytes and sperm into the pelvic cavity
• ICSI
Intracytoplasmic sperm injection, or ICSI, was developed to treat couples who previously had a very poor
probability of achieving fertilization due to the male partner's extremely low numbers of viable sperm.
This treatment, when combined with in vitro fertilization, allows these couples a more favorable probability of
achieving conception.
• TESE
Testicular Sperm Extraction
• MESA
Microsurgical Epididymal Sperm Aspiration