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In-Vitro Cell Technology in India – Origin, Evolution and New frontiers. Dr. Sudarsan Ghosh Dastidar, MD GD Institute for Fertility Research Kolkata, India. HEAL Conference, India International Centre , New Delhi, 23 rd September 2009. Assisted Reproductive Technology (ART) - till now.

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In-Vitro Cell Technology in India – Origin, Evolution and New frontiers

Dr. Sudarsan Ghosh Dastidar, MD

GD Institute for Fertility Research

Kolkata, India

HEAL Conference, India International Centre , New Delhi, 23rd September 2009


Assisted Reproductive Technology (ART) - till now

  • Manipulation of human cells (egg and sperm) out side the body, i.e., “In-Vitro”, started with the breakthrough of test tube baby technology in 1978.
  • Two main modalities –
  • IVF
  • ICSI
  • The advanced laboratory set up and technology required for this may be the basis for embryonic stem cell culture and future cell based therapy.
chronology of ivf icsi research in india
Chronology of IVF-ICSI Research in INDIA
  • 1978 : Dr. Subhas Mukherjee & his team. Birth of Durga.
  • 1982-1986 : Kolkata-Dr. B. N. Chakravorty

Dr. S. Ghosh Dastidar

Birth of Imran.

  • 1983-1986 : Bombay- Dr. I. Hinduja

Dr. Anandkumar

Birth of Harsha ( July 1986 )

  • 1992 : Kolkata- 1st GIFT Baby

Dr. S. Ghosh Dastidar ; Dr. K Ghosh Dastidar

  • 1995 : Kolkata- 1st ICSI ZIFT Baby

Dr. S. Ghosh Dastidar ; Dr. K Ghosh Dastidar

  • 1995 : Mumbai- ICSI Baby

Dr. Firuza Parikh


Dr. Subhas MukherjeeThe pioneer of IVF Research in India.Responsible for inducting me into the emerging field of In-Vitro Fertilization and related science.


World Congress on IVF, Helsinki, Finland, 1984

World Congress on IVF, Istanbul, 2005

Cornell Medical Center, USA

Dr. Bedford and myself, 1987

Prof. Struart Campell, myself and Dr. Kakali Ghosh Dastidar

our initial struggle 1982
Our Initial Struggle (1982)
  • The story of how we developed IVF in India (Kolkata) is one of Herculean struggle. It is necessary to narrate many personal details which are vital to illustrate how breakthroughs in creative science really happen, specially in a country like ours.

Our challenges

    • Non existent infrastructure for IVF related cell culture in India. Hence I had to prepare tissue culture media from scratch, using the bare minimum resources available in Kolkata
    • Frequent power cuts in 1981-82; standby generator was must
    • There was no computer and obviously no internet; thus there was almost no literature/publication/journals available in Kolkata
    • The most challenging job was to develop a tissue culture system which would produce 5% Co2 in air and 95% relative humidity atmosphere since we did not have the modern computerized Co2 incubator.
    • Though it may sound extraordinary to researchers today, one basic challenge was to first identify how the human egg looked under the microscope, BECAUSE THERE WAS NO PHOTOGRAPH/PICTURE available in any book/journal/internet
learning by trial error
Learning by Trial & Error
  • During `81-`82 we had no knowledge even about identification of human egg or the associated laboratory procedures for IVF. Thus we had to start from scratch.
  • I was entrusted with the responsibility to develop basic IVF laboratory aspects like – Preparation of tissue culture media, Oocyte identification; sperm preparation; fertilization; embryo-culture.
  • The only basic knowledge I possessed was of a theoretical nature, and came from my discussions with Late Dr. Subhas Mukherjee.


  • Prof. B. N. Chakraborty was involved in developing the clinical aspects like patient selection, ovarian stimulation and most importantly, oocyte retrieval and finally embryo transfer (ET).
  • I was entrusted to develop the embryo culture laboratory and then the laboratory methods. My major challenges were –
    • To identify human oocyte from follicular fluid aspirate.
    • To device some system to produce 5% CO2 in air atmosphere for IVF and embryo culture – since we did not have modern CO2 incubator.
    • To capacitate spermatozoa – an absolute necessary step to achieve successful fertilization of oocyte.
    • To prepare tissue culture media preparation ; PH and osmolarity control of media. Prof. Subir Dutta, an eminent pathologist of Kolkata extended logistic and technical support.
    • To learn manipulation of human oocyte under stereo microscope.
    • Thus, I was a self trained IVF laboratory expert!!


A typical day

  • Morning 5 – 6 a.m. – cleaning of laboratory, final preparation of media and other lab protocols
  • 7-9 a.m. – basic quality control, preparation for egg recovery.
  • 10:30 a.m. to late evening - I used to carry the test tube containing follicular fluid in a thermos flask to a laboratory in far away Jadavpur University and later, to Indian Statistical Institute, where I was desperately trying to identify the oocytes under stereo microscope (no one in Calcutta then even knew their appearance !)
  • However, soon we realized that in order to achieve pregnancy by IVF we needed a laboratory close to the clinic where oocyte retrieval was being done. Otherwise the temperature and pH of the egg-containing follicular fluid was being altered in this long transit.
  • Thus, I set up Calcutta’s first IVF laboratory (very primitive though!) in my study room at 79/28, A.J.C. Bose Road, Kolkata - 14.
  • Some days I used to finish work at 12 night or even later.

The improvised IVF embryo culture system devised by me in 1983

  • A pre-requisite for IVF is perfectly controlled environment for cell culture, currently provided by state-of-art incubators as shown in the previous slide.
  • During 1982-86 we did not have modern incubator - an absolutely essential equipment for human IVF.
  • Thus I tried to improvise several alternative simple system with locally available materials.
  • Finally I was able to develop a system to produce 5% CO2 in air atmosphere and approximately 85% relative humidity.
improvised embryo culture system developed by me using 5 co 2 in air environment 1982
Improvised embryo culture system developed by me using 5% CO2 in airenvironment (1982)

Modern CO2 Incubators used in our center currently

5% CO2 in air

Test tube containing egg in culture media

Water in petri dish

initial breakthrough in ivf 1982 1984
Initial breakthrough in IVF (1982-1984)
  • After many failures, I achieved fertilization of oocyte and its subsequent cleavage to 6-8 cell stage in June/July `83.
  • Following embryo transfer, a pregnancy occurred. (September `83). Unfortunately, this pregnancy ended in abortion at around 10 weeks.
  • It was a major break through indicating the feasibility of IVF as a clinical method to treat tubal factor infertility.
  • Dr. B. N. Chakraborty and myself jointly presented* this data in 3rd World Congress on IVF, Helsinki, Finland, May 1984.
  • This was the 1st report of successful IVF and embryo transfer resulting in pregnancy in human from India in any World Congress. (May’84)

*Ref. Our Experience of IVF in India; Chakraborty B.N. and Ghosh Dastidar S.; Congress book; 3rd World Congress on IVF, Helsinki, Finland, May 1984

offshoot of ivf technology novel sperm preparation and iui in human infertility
Offshoot of IVF Technology – novel sperm preparation and IUI in human infertility
  • The technology of sperm preparation in IVF, prompted me to venture introducing 100% motile sperm in uterine cavity in patients with unexplained infertility.
  • I was surprised to achieve series of pregnancies following such method during 1982-84.
  • These were the very first few IUI pregnancies in the world.

Abstract of our paper presented at 5th World Congress on Human Reproduction ; Athens, Greece, 1985


ICSI – the major breakthrough in Assisted Reproductive Technology after IVF

  • Till 1993 there was no treatment for severe male factor infertility with very low sperm count/motility, not even by IVF
  • This changed with the advent of Intra Cytoplasmic Sperm Injection (ICSI) which means fertilizing an egg by micro injection with a single viable spermatozoa in the laboratory, first reported in 1992-93 from Brussels, Belgium.
  • I initiated one of India’s first two ICSI programs in Kolkata in January 1994, which was responsible for the birth of a ICSI-ZIFT baby in March 1995.
different stages of fertilized egg and embryo following ivf icsi documented in my lab
Different stages of fertilized egg and embryo following IVF/ICSI documented in my Lab

4 cell stage

PN Stage

8 cell stage

blastocyst embryo
Blastocyst embryo

Morula stage

Blastocyst stage


Human Embryonic Stem Cell (hESC)

  • Stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. For example, a stem cell cannot carry oxygen molecules through the bloodstream (like a red blood cell).
  • Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation.
  • Scientists are just beginning to understand the signals inside and outside cells that trigger each step of the differentiation process.
  • The internal signals are controlled by a cell's genes, which are interspersed across long strands of DNA, and carry coded instructions for all cellular structures and functions.

Historical Landmarks

Scientists discovered ways to derive embryonic stem cells from early mouse embryos


The detailed study of the biology of mouse stem cells led to the discovery of a method to derive stem cells from human embryos and grow the cells in the laboratory. These cells are called human embryonic stem cells.


Major breakthrough - identification of conditions that allow some specialized adult cells to be "reprogrammed" genetically to assume a stem cell-like state. This new type of stem cell, called induced pluripotent stem cells (iPSCs),



How are embryonic stem cells grown in the laboratory

Growing cells in the laboratory is known as cell culture. Human embryonic stem cells are isolated by transferring the inner cell mass from the blastocyst into a plastic laboratory culture dish that contains a nutrient broth known as culture medium.


Pluripotency – the God of small cells

  • Human Embryonic stem cells, as their name suggests, are derived from 4-5 days old surplus human embryos produced by IVF centers.
  • They self-renew indefinitely in the undifferentiated state producing millions of cells.
  • They are pluripotent, which means they can develop into all cells and tissues in the body. Have unique regenerative abilities –
  • The most important potential application – Cell Based Therapies in

1) Diabetes

2) Alzheimer’s disease

3) Spinal cord injury

4) Stroke

5) Heart disease

6) Osteoarthritis

  • With further research it may be possible to understand how cell proliferation is regulated during normal embryonic development or during abnormal cell division and differentiation that leads to cancer.

How are embryonic stem cells stimulated to differentiate?

Directed differentiation of mouse embryonic stem cells.

Diseases that might be treated by transplanting cells generated from human embryonic stem cells include Parkinson's disease, diabetes, traumatic spinal cord injury,Duchenne's muscular dystrophy, heart disease, and vision and hearing loss.


What are the potential uses of human embryonic stem cells and the obstacles that must be overcome before these potential uses will be realized?

  • Studies of human embryonic stem cells will yield information about the complex events that occur during human development.
  • Primary goal - to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs. Scientists know that turning genes on and off is central to this process.
  • Serious medical conditions, such as cancer and birth defects, are due to abnormal cell division and differentiation.
  • A more complete understanding of the genetic and molecular controls of these processes may yield information about how such diseases arise and suggest new strategies for therapy.
  • Testing of drugs - new medications could be tested for safety on differentiated cells generated from human pluripotent cell lines.

Have human embryonic stem cells successfully treated any human diseases?

  • January 23, 2009 - FDA granted clearance for Geron’s (Califormia based pharma company) clinical trial of GRNOPC1 in patients with acute Spinal Cord injury – the World’s 1st Human clinical trial of Embryonic Stem Cell – based therapy
  • Geron’s 2nd HESC product GRNCM1 involves pre clinical trial of cardiomyocytes for treating myocardial disease.

In-Vitro Cell Technology in India – Origin, Evolution and New frontiers



Embryonic Stem Cell from Blastocyst

Cell based therapies

Future regenerative medicine

  • Our human body as the supreme model of omni potent mechanism.
  • It appears that in near future practice of medicine will be rewritten with inclusion of regenerative or restorative medicine.
  • The primitive immortal cells that is embryonic stem cells appears to have the potential to restore the body mechanism from disease process by an unique regenerative approach hitherto unknown to mankind.

E = mc2


enormous energy

Single atom-------------

?Atom Bomb

Regenerative medicine

Millions or trillions of hESC


Embryonic Stem Cell


? Ethical issues


Research Ethics and Stem Cells

  • The ethical question which arises is to what extent should researchers manipulate the most basic processes of human life.
  • However, we need to ask ourselves whether the potential benefits for treatment of disease and improving quality of life outweigh such ethical constraints.
  • Thus, it would seem that research in embryonic stem cell promises much, and also needs guidelines.
  • Here we should keep in mind that during early days of evolution of IVF technology in India, it was precisely the lack of constraining regulation which permitted such exponential growth.