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© 2006 The National Catholic Bioethics Center 443
A Brief History of Human
Diploid Cell Strains
Rene Leiva, M.D.
The Pontifical Academy for Life published a response about the moral legiti-
macy of immunizing children with vaccines manufactured using cell strains derived
from aborted human fetuses. In order to fully appreciate the level of cooperation
involved among different agents, it is important to review the history of the develop-
ment of these cell strains: “The need to articulate a moral reflection on the matter in
question arises mainly from the connection which exists between the vaccines men-
tioned above and the procured abortions from which biological material necessary
for their preparation was obtained.”
1
Human diploid cell strains (HDCSs) are batches of cells that are currently used
for different purposes, including culturing viruses for the manufacturing of vaccines.
HDCS-derived human vaccines have been licensed worldwide for polio IVP and
OVP, rabies, rubella, measles, varicella-zoster, mumps, and hepatitis A. Current vac-
cines contain extremely small traces of the original fetal DNA, while the cell strains
contain the complete fetal chromosomal set. The choice of HDCS was made among
several based on their susceptibility to many human viruses, their good characteriza-
tion, the enormous number of cells obtained from one original culture, their long
1
Pontifical Academy for Life, “Moral Reflections on Vaccines Prepared from Cells
Derived from Aborted Human Fetuses” (June 5, 2005), http://www.academiavita.org/
template.jsp?sez=Documenti&pag=testo/vacc/vacc&lang=English; reprinted in this issue
of the Quarterly on pp. 541–549.
Originally published in the National Catholic Bioethics Quarterly
6.3 (Autumn 2006): 443–451 © 2006 by The National Catholic
Bioethics Center. All rights reserved. Reprinted by permission.
444
THE NATIONAL CATHOLIC BIOETHICS QUARTERLY AUTUMN 2006
storage potential, the low cost of cell procurement, an excellent record of safety, and
the very low risk of latent virus on the cells themselves.
2
Even though there are many cell strains in use in research, the most well known
are WI-38 and MRC-5. These two cell strains come from two deliberately aborted
fetuses. But as the evidence shows, there were more abortions involved to achieve
the technical expertise needed for development of these cell strains. In addition,
other cell strains have been developed for vaccine manufacturing and other pur-
poses. Because of its relevance to this discussion, I will also review the history of the
virus strain RA 27/3, as it is the source of the only rubella vaccine available in North
America and in fact, most of the world. Finally, as my intention is to capture the real
meaning of the evidence, I will quote from the actual sources and personal communi-
cations to try to respect the original meaning.
Human Diploid Cell Strains
The Wistar Institute is a scientific institute located on the campus of the Uni-
versity of Pennsylvania in Philadelphia, specializing in the fields of immunology and
cell biology. Working for the Institute in 1961, Dr. Leonard Hayflick first published a
paper describing twenty-five HDCS: WI-1 through WI-25 (Wistar Institute fetal
samples nos. 1–25). These cell strains were derived from the lung, skin, muscle,
kidney, heart, thyroid, thymus and liver of nineteen separate, electively-aborted fe-
tuses. The purpose of choosing different organs was to test difference in tissue
characteristics. His research included also testing these cell strains’ susceptibility for
different viruses. He stated in this paper that
the isolation and characterization of HDCS from fetal tissue make this type of
cell available as a substrate for the production of live virus vaccines. Other
than the economical advantages, such strains … make the consideration of
their use in the production of human virus vaccine a distinct possibility.
3
Abortion was illegal in the United States at that time, so fetal tissue was pro-
vided by Dr. Sven Gard of the Karolinska Institute Medical School in Stockholm,
Sweden.
4
Dr. Erling Norrby, who later served as chairman of the department of
virology and dean of the medical faculty at the Karolinska Institute, was a graduate
student there during this period. He dissected many of the aborted fetuses:
My predecessor as professor of virology at the Karolinska Institute in
Stockholm, Sven Gard, spent a sabbatical year at the Wistar Institute in 1959,
2
M. A. Fletcher, L. Hessel, and S. A. Plotkin, “Human Diploid Cell Strains (HDCS)
Viral Vaccines,” Developments in Biological Standardization 93 (1998): 97–107;
L. Hayflick, “History of Cell Substrates Used for Human Biologicals,” Developments in
Biological Standardization 70 (1989): 11–26.
3
L. Hayflick and P. S. Moorhead, “The Serial Cultivation of Human Diploid Cell
Strains,” Experimental Cell Research 25.3 (December 1961): 618.
4
E. Norrby, “Listen to the Music: The Life of Hilary Koprowski (review),” Perspec-
tives in Biology and Medicine 44.2 (Spring 2001): 304; Fletcher, Hessel, and Plotkin,
“Human Diploid Cell Strains,” 97–98.
445
LEIVA HUMAN DIPLOID CELL STRAINS
two years after the institution had been taken over by the dynamic Koprowski.
One of my duties as a young student in the laboratory in Stockholm was to
dissect human fetuses from legal abortions and send organs to the Wistar In-
stitute. Such material was the source of many important studies of cell lines at
the Institute, such as Leonard Hayflick’s study of WI-38 cells.
5
Hayflick and his collaborators (including Anthony Girardi from the Merck Institute
for Therapeutic Research) started working with these cell strains to develop viral
vaccines: a poliovirus vaccine was developed in the WI-1 cell strain in 1962. By this
time, fifty HDCSs had been made. Finally, after these improvements in the tech-
nique, Hayflick published his reports of the development of WI-38.
6
WI-38 was
obtained from a three-month-old female fetus:
This fetus was chosen by Dr. Sven Gard, specifically for this purpose. Both
parents are known, and unfortunately for the story, they are married to each
other, still alive and well, and living in Stockholm, presumably. The abortion
was done because they felt they had too many children. There were no familial
diseases in the history of either parent, and no history of cancer specifically in
the families.
7
This report also mentions two additional cell strains: WI-26 from a male fetus (lung)
and WI-44 from a female fetus (lung). Both fetuses were about three-months’ gesta-
tion as well.
8
An article co-authored by Gard and colleagues at the Wistar Institute stated, in
reference to Hayflick’s cell strains, that
a human diploid cell strain derived from a fetal lung tissue was employed in-
stead of monkey-kidney cells for the preparation of the attenuated poliovirus
vaccine utilized in our study. The cell strain, cultivated especially for the pro-
duction of virus vaccines, retains relatively constant morphology and chromo-
somal characteristics … and it is believed to be free of all known adventitious
agents. The expectation is that cells originating from a single fragment of
tissue, passages of which are stored and cultivated at will, could be used in
place of monkey cells … to make large quantities of vaccine.
9
On an interesting note, Hayflick was concerned about the continued capture of
wild monkeys and their existence as species and saw HDCS as a solution to this
5
Norrby, “Listen to the Music,” 304.
6
L. Hayflick, “The Limited In Vitro Lifetime of Human Diploid Cell Strains,” Ex-
perimental Cell Research 37 (March 1965): 614–636; L. Hayflick et al., “Preparation of
Poliovirus Vaccines in a Human Fetal Diploid Cell Strain,” American Journal of Hygiene
75 (March 1962): 240–258.
7
“Gamma Globulin Prophylaxis; Inactivated Rubella Virus; Production and Biologics
Control of Live Attenuated Rubella Virus Vaccines” [no author given], American Journal
of Diseases of Children 118.2 (August 1969): 377–278.
8
Hayflick et al., “Preparation of Poliovirus Vaccines,” 240, 244, 254.
9
J. S. Pagano et al., “The Response and the Lack of Spread in Swedish School Chil-
dren Given an Attenuated Poliovirus Vaccine Prepared in a Human Diploid Cell Strain,”
American Journal of Hygiene 79 (January 1964): 74–75.
446
THE NATIONAL CATHOLIC BIOETHICS QUARTERLY AUTUMN 2006
problem. A previous ethical version of these vaccines was developed from the kidney
cells of the African green monkeys, an endangered species.
10
Also, Hayflick himself
became a vaccine developer for a polio vaccine and fought and won the legal right to
hold a patent and profit from WI-38.
11
Finally, Hayflick was one of the co-signers of
a letter sent to President Bush in 2001 to support the destruction of human embryos
that occurs in embryonic stem cell research:
For the past thirty-five years many of the common human virus vaccines—
such as measles, rubella, hepatitis A, rabies and poliovirus—have been pro-
duced in cells derived from a human fetus to the benefit of tens of millions of
Americans. Thus precedent has been established for the use of fetal tissue that
would otherwise be discarded.
12
He is on the scientific advisory board of Advanced Cell Technology, the private
company that claimed to have cloned the first human embryo in 2002.
Dr. J. P. Jacobs published the development of the cell strain MRC-5 (Medical
Research Council strain no. 5) in 1970. He replicated Hayflick’s work with the
purpose of creating cells strains for the production of vaccines:
The stability and integrity of the human foetal cell strain WI-38 … explain the
value of such material for the isolation of viruses and in the development of
vaccines. We have developed another strain of cells, also derived from fetal
lung tissue, taken from a fourteen-week male fetus removed for psychiatric
reasons from a twenty-seven-year-old woman with a genetically normal fam-
ily history and no sign of neoplastic disease both at abortion and for at least
three years afterwards.
13
There is the possibility that there may have been previous abortions performed to
create MRC-5. In fact, Jacobs reported creating a second cell strain, MRC-9, by the
use of a different aborted fetus:
the cells were derived from the lungs of a female fetus in 1974, whose gesta-
tional age was about fifteen weeks. The fetus was of normal development and
was delivered of a fourteen-year-old mother whose pregnancy was terminated
by therapeutic abortion because she was unmarried. The medical history of the
mother and her family indicated nothing abnormal according to information
given by the gynecologist who performed the operation. The lungs were dis-
sected from the fetus immediately following the abortion…
14
10
L. Hayflick, “The Choice of the Cell Substrate for Human Virus Vaccine Produc-
tion,” Laboratory Practice 19.1 (January 1970): 59.
11
L. Hayflick, “History of Cell Substrates Used for Human Biologicals,” Develop-
ments in Biological Standardization 70 (1989): 15.
12
K. J. Arrow et al., “Nobel Laureates’ Letter to President Bush,” Washington Post,
February 22, 2001, A02.
13
J. P. Jacobs, C. M. Jones, and J. P. Baille, “Characteristics of a Human Diploid
Cell Designated MRC-5,” Nature 227.5254 (July 11, 1970): 168.
14
J. P. Jacobs, A. J. Garrett, and R. Merton, “Characteristics of a Serially Propagated
Human Diploid Cell Designated MRC-9,” Journal of Biological Standardization 7.2 (April
1979): 114.
447
LEIVA HUMAN DIPLOID CELL STRAINS
Newer HDCSs continued to be made as back-ups for the current cell strains.
Among the most common ones are IMR-90, cell strain 293, and PER C6.
15
In short,
IMR-90 was established from a sixteen-week-old human fetus on July 7, 1975, from
a therapeutic abortion performed on a thirty-eight-year-old white mother of six.
16
Cell strain 293 was made from human embryonic kidney cells from an aborted fetus
in 1972, and cell strain PER C6 from human embryonic retina cells from an abortion
in 1985. The main researcher was Dr. A. J. van der Eb at Leiden University in
Holland. Van der Eb dissected the fetuses himself, which were healthy. PER C6
came from an eighteen-week-old aborted fetus because “the woman wanted to get
rid of the fetus and the father was unknown.” Van der Eb stated that “PER C6 was
made just for the pharmaceutical manufacturing of adenovirus vectors.” He also
added, “I realize that this sounds a bit commercial, but PER C6 was made for that
particular purpose.” Cell strain 293 was made for “basic research.”
17
At least fifty
companies have licensed PER C6, including Merck, the sole manufacturer of the
only rubella vaccine available in North America.
18
The Origin of Rubella Virus RA 27/3
Currently, the virus strain (RA 27/3) found in the rubella vaccine most com-
monly used around the world was developed by Dr. Stanley Plotkin and colleagues
at the Wistar Institute.
19
The RA 27/3 (rubella abortus, twenty-seventh fetus, third
tissue extract) virus strain was obtained from a female human fetus in a series of
twenty-seven abortions in the United States: “Explant cultures were made of the
dissected organs of a particular fetus aborted because of rubella, the twenty-seventh
in our series of fetuses aborted during the 1964 epidemic.”
20
“This fetus was from a
twenty-five-year-old mother exposed to rubella eight weeks after her last menstrual
period…. The fetus was surgically aborted seventeen days after maternal illness and
dissected immediately… It was then grown on WI-38.”
21
15
W. W. Nichols et al., “Characterization of a New Human Diploid Cell Strain, IMR-
90,” Science 196.4285 (April 1, 1977): 60; FDA Vaccines and Related Biological Products
Advisory Committee, transcript of meeting May 16, 2001, “Session on Designer Cell Sub-
strate,” http://www.fda.gov/ohrms/dockets/ac/01/transcripts /3750t1_01.pdf.
16
Nichols et al., “Characterization of a New Human Diploid Cell Strain,” 60.
17
Alex J. van der Eb, in “Session on Designer Cell Substrates,” FDA meeting tran-
script, May 16, 2001.
18
L. Xie et al., “Large-Scale Propagation of a Replication-Defective Adenovirus Vec-
tor in Stirred-Tank Bioreactor PER.C6 Cell Culture under Sparging Conditions,” Biotech-
nology and Bioengineering 83.1 (July 5, 2003): 45.
19
S. A. Plotkin, D. Cornfeld, and T.H. Ingalls, “Studies of Immunization with Living
Rubella Virus: Trials in Children with a Strain Cultured from an Aborted Fetus,” American
Journal of Diseases of Children 110.4 (October 1965): 381–382.
20
S. A. Plotkin et al., “Attentuation of RA 27-3 Rubella Virus in WI-38 Human Dip-
loid Cells,”American Journal of Disabilities of Children 118.2 (August 1969): 178.
21
Plotkin, Cornfeld, and Ingalls, “Studies of Immunization with Living Rubella Vi-
rus,” 381–382.
448
THE NATIONAL CATHOLIC BIOETHICS QUARTERLY AUTUMN 2006
The new vaccine was tested on children at a Roman Catholic orphanage in
Philadelphia. It is documented that there were other effective virus strains already
made at that time which had been obtained from other non-abortion-related meth-
ods.
22
Nevertheless, the researchers seem to have chosen RA 27/3 because of its
lack of contaminants, immunogenicity, low side effects, and enormous cell growth.
Also, RA 27/3 was further cultured with the cell strain WI-38.
Additionally, six months after publishing this research, Plotkin and colleagues
published an article documenting forty, not twenty-seven, abortions:
Out of the forty rubella fetuses cultured, cell strains were derived from thirty-
four; in most cases they originated from pieces of skin and muscle obtained at
curettage … rubella virus was isolated from the supernatant culture medium
of cell strains derived from eighteen fetuses; sixteen fetuses yielded cell strains
which were rubella negative.
23
The RA 27 fetus was not the first fetus in the series to be positive for rubella virus. It
is not clear why they continued with the series.
Later, Drs. J. Hoskins and Plotkin tested the action of the RA 27/3 virus on
different systems of human embryonic cell cultures. Additional cell strains were
made from more fetuses originating in both elective abortions (twenty-one) and
miscarriages (seven):
24
Two groups of human fetuses, eight to twenty weeks, were used for the initia-
tion of diploid cell strains. The first group consisted of normal embryos ob-
tained by hysterotomy and flown from Scandinavia.… The second group rep-
resented spontaneous abortions obtained from the gynecologic service of the
Philadelphia General Hospital.
25
From the records, it also seems that both sources of cell strains yielded similar
efficacy:
Cell strains derived from twenty-nine fetuses were examined. Twenty one of
these originated from surgical abortions, while seven came from spontane-
ously aborted fetuses. One cell strain was of uncertain origin. At the start of
these studies, most importance was attached to HDCS derived from the surgi-
cally aborted fetuses since these could be presumed to be normal. In fact, no
differences in any of the parameters studied could be found between the two
groups of fetuses, and no distinction will henceforth be made between them.
26
22
F. T. Perkins, “Licensed Vaccines,” Reviews of Infectious Diseases 7 (March–April
1985), Suppl 1: S73–S76.
23
T. H. Chang et al., “Chromosome Studies of Human Cells Infected in Utero and In
Vitro with Rubella Virus,” Proceedings of the Society for Experimental Biology and Medi-
cine 122.1 (May 1966): 237–238.
24
J. M. Hoskins and S. A. Plotkin, “Behaviour of Rubella Virus in Human Diploid Cell
Strains. I. Growth of Virus,” Archiv fur die Gesamte Virusforschung 21.3 (1967): 285; J.
M. Hoskins and S. A. Plotkin, “Behaviour of Rubella Virus in Human Diploid Cell Strains. II.
Studies of Infected Cells,” Archiv fur die Gesamte Virusforschung 21.3 (1967): 297.
25
Hoskins and Plotkin, “Behaviour of Rubella Virus I,” 285.
26
Hoskins and Plotkin, “Behaviour of Rubella Virus II,” 297.
449
LEIVA HUMAN DIPLOID CELL STRAINS
(Please note the arithmetic error, as twenty-one and seven do not add up to twenty-
nine fetuses. It could have been one more aborted fetus or one extra miscarriage).
Plotkin later developed experimental polio, varicella, and cytomegalovius vac-
cines. He is now employed at Sanofi Pasteur, a vaccine manufacturer. He believes
that his rubella vaccine has helped to prevent many abortions: “‘I have no doubt that
rubella vaccination has prevented thousands and thousands of abortions,’” he said.
‘From strictly an arithmetical assessment, the good done by the vaccine—if you are
opposed to abortion—is infinitely greater than any possible harm.’”
27
The Manufacturers
At this point, it is important to mention that pharmaceutical companies in both
Europe and North America quickly became involved in the use of HDCSs.
28
The
World Health Organization in joint effort with the Wistar Institute funded meetings
and training sessions with individuals interested in learning about HDCSs during the
1960s.
29
Given the fact that the research was public knowledge, it is impossible that the
companies were unaware of the ethical predicament. To the researchers’ credit, they
never hid the real source of the cells, as the titles of their articles confirm.
30
As stated
in the written evidence, at least one collaborator in the research was working for a
pharmaceutical company at the time the research was being done. Besides, the mini-
mum requirements of safety standards dictate that a manufacturing pharmaceutical
company know in detail the source of its raw material. The question has been posed
whether this is like benefiting from the use of an organ from an executed persons or
from unethical Nazi research.
31
The Abortions
As I am also preparing an article for a Canadian medical journal on immuniza-
tion, refusal, safety, and informed consent but not necessarily on the moral point of
view, I needed to be able to assess the vaccines’ track of safety. In order to do this, it
was necessary to trace back to the original abortions to ensure that there were no
foreign dangerous contaminants. I thus emailed Dr. Norrby about this problem. Dr.
Norrby stated that the cell strains were safe, since the tissue was collected in a very
sterile manner:
When we collected the organs this was done immediately after the legal abor-
tion. We were on duty to immediately perform the sampling and to arrange for
27
D. Brown, “Rubella Virus Eliminated in the United States,” Washington Post,
March 21, 2005, A07.
28
Fletcher, Hessel, and Plotkin, “Human Diploid Cell Strains,” 97–98.
29
Hayflick, “History of Cell Substrates,” 15.
30
Plotkin, Cornfield, and Ingalls, “Studies of Immunization with Living Rubella Virus,”
381–382.
31
R. K. Zimmerman, “Ethical Analyses of Vaccines Grown in Human Cell Strains De-
rived from Abortion: Arguments and Internet Search,” Vaccine 22.31–32 (October 22,
2004): 4238–4244.
450
THE NATIONAL CATHOLIC BIOETHICS QUARTERLY AUTUMN 2006
an as rapid transport as possible over the Atlantic Ocean. The fetal material
arrived by car from the nearby hospital to our laboratory enwrapped in a green
surgical cloth. Maximal sterility was critical to allow an outgrowth of fetal
cells without any contamination after the transport under cold conditions to
the Wistar Institute.
32
Whether there was any coercion in the abortions in order to procure these cell strains
is unknown. We will also probably never know whether the mothers were actually
aware that their abortions may have been used for the creation of cell strains, given
what Dr. Norrby states regarding informed consent:
Remember that at the time in the early 1960s when organs from aborted fe-
tuses were collected and sent to the Wistar Institute no one had as yet invented
the concept of informed consent. I am absolutely convinced that there is no
remaining documentation about the fetuses used from the Department of Vi-
rus Research of the Karolinska Institute at the time. I was the head of this
department between 1972 and 1997. Thus in case there is no documentation
that allows identification of fetal samples at the Wistar Institute, there is no
way of tracing them. I do in fact remember the time well, because we as gradu-
ate students made the dissections collecting organs.
33
Conclusion
There is clear evidence that research around the development of the RA 27/3
rubella vaccine included the performance and coordination of at least eighty abor-
tions, including the two individual abortions for the creation of WI-38 and RA 27/3.
Development of MRC-5 used one abortion, but there is a strong indication that more
abortions occurred. Evidence also seems to indicate that there was intention in the
act of utilizing abortions for the creation of cell strains, most likely because the tissue
source ensures an absence of contamination and a high growth titer. There have been
other abortions as a result of the need to create more cell strains for use in vaccine
development.
34
Pharmaceutical companies are actively involved in this research and
32
E. Norrby, e-mail response to a message from R. Leiva on January 23, 2006. Dr. Leiva
had asked: “You mention that the step you were involved in (dissection of the fetal tissue) was
done under sterile conditions. What about the steps of the procedure prior to that? Do you know
anything about the conditions between the therapeutic abortions and the dissections? Were they
both happening one after the other in the same facility and laboratory standards?”
33
E. Norrby, e-mail response to a message from R. Leiva on January 20, 2006. Dr. Leiva
had asked: “(1) Was the reason for the pregnancies’ termination medical or socio-thera-
peutic? (i.e., were diseases in the fetuses the reasons for the terminations?) (2) Was there
good documentation regarding the health of parents of the fetuses? If so, where this can be
obtained? (3) How were particular fetuses chosen? (Were there any medical reasons for
choosing a particular fetus as Dr. Gard says in reference 2, or did the parents have any in-
put in the choice. And (4) How was the termination–dissection–set-up organized to de-
crease the risk of introducing any kind of contaminants?”
34
M. G. Pau et al., “The Human Cell Line PER.C6 Provides a New Manufacturing System
for the Production of Influenza Vaccines,” Vaccine 19.17–19 (March 21, 2001): 2716–2721.
451
LEIVA HUMAN DIPLOID CELL STRAINS
new vaccines are being made with unethical cell strains.
35
There are alternative
ethical viral vaccines already made with modern cell substrates: Cell lines such as
mammalian cells like Vero monkey cells and Chinese hamster ovary cells (e.g. some
Polio IVP).
36
Alternatively, producing vaccines with antigens using recombinant DNA
technology is another option (e.g.: hepatitis B).
37
Efforts should be made to encour-
age research on these and other novel ethical sources.
35
M. N. Oxman et al. for the Shingles Prevention Study, “A Vaccine to Prevent Her-
pes Zoster and Postherpetic Neuralgia in Older Adults,” New England Journal of Medi-
cine 352.22 (June 2, 2005): 2271–2284.
36
L. Hayflick, “History of Cell Substrates,” 24.
37
D. B. Huang, J. J. Wu, and S. K. Tyring, “A Review of Licensed Viral Vaccines, Some
of their Safety Concerns, and the Advances in the Development of Investigational Viral Vac-
cines,” Journal of Infection 49.3 (October 2004): 179–209.