Jack Bley, DVM

A “Best of Journal Club” presentation!!

ETHICAL ISSUES

• ABORTED FETUSES AS SOURCES OF CELL LINES

• INFORMED CONSENT/RESEARCH ON MINORS

• RUBELLA-INFLUENCED ABORTIONS

• FETUS 27 KIDNEY CELLS

• CAUSING INFECTION TO STUDY INFECTION

• STOPPING THE NEXT EPIDEMIC

• ANIMALS IN BIOMEDICAL RESEARCH

• ANTI-VACCINERS

Leonard Hayflick

b 1928 in Philly

Mycoplasma

Hayflick’s medium

WI – 38

Cell Associates Inc.

Stanford U & UCSF

NIH battles

Aging research

Experimental Cell Research 25, 585-621 (1961) p585

THE SERIAL CULTIVATION OF HUMAN DIPLOID CELL STRAINS

L. HAYFLICK and P. S. MOORHEAD

Wistar Institute of Anatomy and Biology, Philadelphia, Pa., U.S.A.

Received May 15, 1961

This paper is a “Citation Classic” because other papers refer to it so frequently: 4469 citations and 60 references in the literature.

WHAT ARE DIPLOID CELLS?

Hayflick L, Moorhead PS.. The serial cultivation of human diploid cell strains. Exp Cell Res 25: 585-621.

Abstract:

The isolation and characterization of 25 strains of human diploid fibroblasts derived from fetuses are described. Routine tissue culture techniques were employed. Other than maintenance of the diploid karyotype, ten other criteria serve to distinguish these strains from heteroploid cell lines. These include retention of sex chromatin, histotypicaldifferentiation, inadaptability to suspended culture, non-malignant characteristics in vivo, finite limit of cultivation, similar virus spectrum to primary tissue, similar cell morphology to primary tissue, increased acid production compared to cell lines, retention of Coxsackie A9 receptor substance, and ease with which strains can be developed. Survival of cell strains at − 70 °C with retention of all characteristics insures an almost unlimited supply of any strain regardless of the fact that they degenerate after about 50 subcultivations and one year in culture. A consideration of the cause of the eventual degeneration of these strains leads to the hypothesis that non-cumulative external factors are excluded and that the phenomenon is attributable to intrinsic factors which are expressed as senescence at the cellular level. With these characteristics and their extremely broad virus spectrum, the use of diploid human cell strains for human virus vaccine production is suggested. In view of these observations a number of terms used by cell culturists are redefined.

An international leader in biomedical science, with special expertise in oncology, immunology, infectious disease and vaccine research. Located in the University City section of Philadelphia, Wistar was founded in 1892 as America's first nonprofit institution solely focused on biomedical research and training, it works to ensure that research advances from the laboratory to the clinic as quickly as possible. Known worldwide for vaccinedevelopment, some of the Institute's accomplishments are its contributions to the creation of vaccines for rubella (German Measles), rotavirus and rabies.

The Wistar rat is an outbred albino rat. This breed was developed at the Wistar Institute in 1906.

Louis Pasteur, 19th Century,

French microbiologist

16 WEEK OLD FETUS

WI-38

WI-38 was invaluable to early researchers, especially those studying virology and immunology, since it was a readily-available cell line of normal human tissue, unlike the HeLa line, which were cancerous cells. Researchers in labs across the globe have since used WI-38 in their discoveries, most notably in the development of vaccines. Over a billion vaccine doses worldwide can be

traced to work done on WI-38: measles, rabies, adeno, chickenpox, polio, rubella.

Organism Homo sapiens, human

Tissue lung

Cell Type fibroblast

Product Format frozen

Morphology fibroblast

Biosafety Level

1 Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Age 3 months gestation fetus

Gender female

Ethnicity Caucasian

Applications This cell line can be used for virucidetesting.

WI-38 (ATCC® CCL-75™)

ATCC medium: 1463 Modified Hayflick medium

• Heart Infusion Broth (BD 238400)........28.5 g

• Agar, Noble (BD 214230), if necessary....9.6 g

• Distilled water........................900.0 ml

• Autoclave at 121OC for 15 minutes. Cool to 50OC and add aseptically:

• Horse serum (normal)...................200.0 ml

• Yeast Extract Solutuion (GIBCO 18180)..100.0 ml

• 0.2% Calf Thymus DNA (Sigma D1501)......12.0 ml

• Adjust medium to pH 7.8.

Another Classic Paper

Congenital cataract following German measles in the mother

N. McAlister GreggAustralia, 1941

CONGENITAL RUBELLA SYNDROME

Stanley Plotkinb 1932 in the Bronx

https://media.historyofvaccines.org/mobile/video/320/999001.mp4

Rubella = German Measles

There was a pandemic of rubella between 1962 and 1965, starting in Europe and spreading to the United States.

In the years 1964–65, the United States had an estimated 12.5 million rubella cases.

This led to 11,000 miscarriages or therapeutic abortions and 20,000 cases of congenital rubella syndrome. Of these, 2,100 died as neonates, 12,000 were deaf, 3,580 were blind, and 1,800 were mentally retarded. In New York alone, CRS affected 1% of all births.

In 1969 a live attenuated virus vaccine was licensed. In the early 1970s, a triple vaccine containing attenuated measles, mumps and rubella (MMR) viruses was introduced.

By 2006, confirmed cases in the Americas had dropped below 3000 a year.

Rubella is the third disease to be eradicated from the western hemisphere with vaccination after smallpox and polio.

VIRAL VACCINES IN 1961

• POLIO – monkey kidneys

• RABIES – rabbit spinal cord

• SMALLPOX – calf skin

• FOOT AND MOUTH DISEASE

• HOG CHOLERA

• SALK - 1955

• SABIN – 1961

• GROWN ON MONKEY KIDNEY CELLS

In addition to his work on the Polio vaccine, Koprowski (along with Stanley Plotkin and Tadeusz Wiktor) did significant work on an improved vaccine against rabies. The group

developed the HCDV rabies vaccine (WI-38 cells) in the 1960s at the Wistar Institute. It was licensed for use in the United States in 1980.

https://www.webofstories.com/play/leonard.hayflick/143

In addition to his work on the Polio vaccine, Koprowski (along with Stanley Plotkin and Tadeusz Wiktor) did significant work on an improved vaccine against rabies. The group

developed the HCDV rabies vaccine (WI-38 cells) in the 1960s at the Wistar Institute. It was licensed for use in the United States in 1980.

https://www.webofstories.com/play/leonard.hayflick/144

Maurice Ralph Hilleman (1919–2005)While working on improving Merck's commercially available polio vaccine in 1960, Hilleman uncovered that a cancer-causing virus, SV40, was present in the polio vaccine. SV40 was a virus found in both monkeys and humans and could cause tumors to grow. Hilleman found that SV40 was already present in the monkey kidney cells in which Merck researchers grew weakened versions of poliovirus. When they tried to isolate the weakened polio virus, they also accidentally isolated SV40, both of which they included in the vaccine. Hilleman's findings caused Merck to remove its polio vaccine from the market and led the US Food and Drug Administration (FDA), headquartered in Silver Spring, Maryland, in 1963 to establish screening programs looking for other stray viruses in vaccines. Although researchers did not find links between monkey viruses like SV40 and cancer in humans, Hilleman's findings convinced vaccine researchers to use human cells to develop vaccines.

Experimental Biology and Medicine

The Vacuolating Virus, S.V.40

B. H. Sweet, M. R. Hilleman

First Published November 1, 1960

WHAT’S WRONG WITH THIS PICTURE?

MONKEY SAFETY TEST FOR POLIO VACCINES

THE USE OF ANIMALS IN BIOMEDICAL RESEARCH

ETHICAL ISSUES

• ABORTED FETUSES AS SOURCES OF CELL LINES

• INFORMED CONSENT

• RUBELLA ABORTIONS

• FETUS 27

• CAUSING INFECTION TO STUDY INFECTION

• STOPPING THE NEXT EPIDEMIC

• ANTI-VACCINERS

• ANIMALS IN BIOMEDICAL RESEARCH

Telomeres and telomerase: the path from

maize, Tetrahymena

and yeast to human cancer and aging

Elizabeth H Blackburn, Carol W Greider & Jack W Szostak

NATURE MEDICINE

VOLUME 12 |

NUMBER 10 |

OCTOBER2006

4/27/2004 5/3/2004 AR 20 M (organ donor)

Bat, Tb

5/25/200 5/31/2004 OK 53 MLiver transplant

Bat, Tb

5/29/2004 6/9/2004 TX 50 FKidney transplant

Bat, Tb

6/2/2004 6/10/2004 TX 55 FArterial transplant

Bat, Tb

5/27/2004 6/21/2004 TX 18 MKidney transplant

Bat, Tb

10/12/2004

Survived WI 15 F Bite Bat, unknown

10/19/2004

10/26/2004

CA 22 MUnknown-El Salvador

Dog, El Salvador

9/27/2005 9/27/2005 MS 10 M Contact Bat, unknown

5/4/2006 5/12/2006 TX 16 M Contact Bat, Tb

9/30/2006 11/2/2006 IN 10 F Bite Bat, Ln

11/15/2006

12/14/2006

CA 11 MBite-Philipines

Dog, Philipines

9/19/200710/20/2007

MN 46 M Bite Bat, unknown

3/16/2008 3/18/2008 CA 16 MBite-Mexico

Fox, Tb-related

11/19/2008

11/30/2008

MO 55 M Bite Bat, Ln

2/25/2009 Survived TX 17 F Contact Bat, unknown

10/5/200910/20/2009

IN 43 M Unknown Bat, Ps

10/23/2009

11/20/2009

VA 42 MContact-India

Dog, India

10/20/2009

11/11/2009

MI 55 M Contact Bat, Ln

8/2/20108/21/2010

LA 19 MBite-Mexico

Bat, Dr

12/24/2010

1/10/2011 WI 70 M Unknown Bat, Ps

4/30/2011 Survived CA 8 F Unknown Unknown

6/30/2011 7/20/2011 NJ 73 F Bite-Haiti Dog, Haiti

8/14/2011 8/21/2011 NY 25 MContact-Afghanistan

Dog, Afghanistan

9/1/2011 8/21/2011 NC 20 M Unknown Raccoon, eastern US

9/1/201110/14/2011

MA 40 MContact-Brazil

Dog, Brazil

12/3/201112/19/2011

SC 46 F Unknown Tb

12/22/2011

1/23/2012 MA 63 M Contact My Sp

7/31/2012 7/6/2012 CA 34 M Bite Bat, Tb

5/16/2013 6/11/2013 TX 28 MUnknown-Guatemala

Dog, Guatemala