U.S. patent application number 10/668179 was filed with the patent office on 2004-04-01 for methods for regulating the specific lineages of cells produced in a human hematopoietic cell culture, methods for assaying the effect of substances of lineage-specific cell production, and cell compositions produced by these cultures.
This patent application is currently assigned to The Regents of the University of Michigan. Invention is credited to Armstrong, R. Douglas, Clarke, Michael F., Emerson, Stephen G., Palsson, Bernhard O..
Application Number | 20040063201 10/668179 |
Document ID | / |
Family ID | 27541300 |
Filed Date | 2004-04-01 |
United States Patent
Application |
20040063201 |
Kind Code |
A1 |
Palsson, Bernhard O. ; et
al. |
April 1, 2004 |
Methods for regulating the specific lineages of cells produced in a
human hematopoietic cell culture, methods for assaying the effect
of substances of lineage-specific cell production, and cell
compositions produced by these cultures
Abstract
Methods, including culture media conditions, which provide for
in vitro human stem cell division and/or the optimization of human
hematopoietic progenitor cell cultures and/or increasing the
metabolism or GM-CSF secretion or IL-6 secretion of human stromal
cells and/or a method for assaying the effect of a substance or
condition on a human hematopoietic cell population, and/or
depleting the malignant cell or T-cell and B-cell content of a
human hematopoietic cell population are disclosed. The methods rely
on culturing human stem cells and/or human hematopoietic progenitor
cells and/or human stromal cells in a liquid culture medium which
is replaced, preferably perfused, either continuously or
periodically, at a rate of 1 ml of medium per ml of culture per
about 24 to about 48 hour period, and removing metabolic products
and replenishing depleted nutrients while maintaining the culture
under physiologically acceptable conditions. Optionally, growth
factors are added to the culture medium. The disclosed culture
conditions afford improved methods for bone marrow
transplantation.
Inventors: |
Palsson, Bernhard O.; (La
Jolla, CA) ; Armstrong, R. Douglas; (Ann Arbor,
MI) ; Clarke, Michael F.; (Ann Arbor, MI) ;
Emerson, Stephen G.; (Wayne, MI) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
The Regents of the University of
Michigan
Ann Arbor
MI
|
Family ID: |
27541300 |
Appl. No.: |
10/668179 |
Filed: |
September 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10668179 |
Sep 24, 2003 |
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08857594 |
May 16, 1997 |
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6667034 |
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08857594 |
May 16, 1997 |
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08334011 |
Nov 2, 1994 |
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5635386 |
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08334011 |
Nov 2, 1994 |
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07815513 |
Jan 2, 1992 |
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07815513 |
Jan 2, 1992 |
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07740590 |
Aug 5, 1991 |
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5399493 |
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07740590 |
Aug 5, 1991 |
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07737024 |
Jul 29, 1991 |
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07737024 |
Jul 29, 1991 |
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07628343 |
Dec 17, 1990 |
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07628343 |
Dec 17, 1990 |
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07366639 |
Jun 15, 1989 |
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Current U.S.
Class: |
435/366 ;
435/370; 435/372 |
Current CPC
Class: |
C12N 5/0647 20130101;
C12N 2500/34 20130101; C07K 14/5412 20130101; C07K 14/705 20130101;
C12N 15/85 20130101; C12N 2740/10043 20130101; C12N 2501/39
20130101; C12N 2501/23 20130101; C12N 15/86 20130101; C12N 2501/125
20130101; C07K 14/5403 20130101; C12N 2501/22 20130101; C07K 14/535
20130101; C12N 5/0641 20130101; C12N 2502/99 20130101; C12N 5/0642
20130101; C12N 2501/14 20130101; C12N 2502/1394 20130101; A61K
48/00 20130101 |
Class at
Publication: |
435/366 ;
435/372; 435/370 |
International
Class: |
C12N 005/08 |
Claims
What is claimed as new and desired to be secured by Letters Patent
of the United States is:
1. A method for controlling the lineage development in an in vitro
human tissue system, comprising culturing human stem and/or
progenitor cells in a liquid culture medium which is replaced at a
rate of about 1 ml of medium per 1 ml of culture per about 24 to
about 48 hours, and removing metabolic products and replenishing
depleted nutrients, while maintaining said culture under
physiologically acceptable conditions, and adjusting the
concentration of hematopoietic growth factors to select for
enhanced production of a desired cell type.
2. The method of claim 1, wherein at least one member selected from
the group consisting of human peripheral blood mononuclear cells,
human bone marrow cells, human fetal liver cells, human cord blood
cells, human spleen cells and mixtures thereof are cultured.
3. The method of claim 1, wherein said cells comprise human stromal
stem and/or progenitor cells and/or mature stromal cells.
4. The method of claim 1, wherein said cells comprise human bone
marrow stromal cells.
5. The method of claim 1, wherein B-cells and T-cells are depleted
and said medium is substantially free of IL-2.
6. The method of claim 1, wherein active and prolonged
erythropoiesis is achieved by adding IL-3 and Epo to said
medium.
7. The method of claim 6, wherein GM-CSF is added to said
medium.
8. The method of claim 1, wherein active and prolonged
granulopoiesis is achieved by adding IL-3 and GM-CSF to said
medium.
9. The method of claim 1, wherein malignant cells are depleted.
10. The method of claim 1, wherein said human cells comprise
genetically transformed stem and/or progenitor cells.
11. The method of claim 1, wherein said medium is replaced
continuously.
12. The method of claim 11, wherein replacement of said medium
comprises perfusing fresh medium through at least part of the mass
of said human stem cells.
13. The method of claim 1, wherein said medium is replaced
periodically or intermittently.
14. The method of claim 13, wherein replacement of said medium
comprises perfusing fresh medium through at least part of the mass
of said human cells.
15. The method of claim 1, wherein said medium comprises animal or
human sera or plasma.
16. The method of claim 1, wherein said medium is substantially
serum free.
17. The method of claim 1, wherein said media comprises a
corticosteroid.
18. The method of claim 1, comprising maintaining glucose
concentration in said medium in the range of from 5 to 20 mM,
lactate concentration in said medium below about 35 mM, glutamine
concentration in said medium in the range of from 1 to 3 mM, and
ammonia concentration in said medium below 2.4 mM.
19. The method of claim 1, further comprising removing nonadherent
cells continuously, periodically, or intermittently, without
distubing adherent cells.
20. A method for assaying the effect of a substance or physical
condition on a human hematopoietic cell mass, comprising culturing
a first portion of said human hematopoietic cell mass, including
dividing human stem cells and progenitor cells, in a liquid culture
medium which is replaced, either continuously, periodically, or
intermittently, at a rate of about 1 ml of medium per ml of culture
per about 24 to about 48 hour period in the presence of said
substance, and removing metabolic products and replenishing
depleted nutrients while maintaining said culture under
physiologically acceptable conditions, and comparing the
compositional profile of the human hematopoietic cell mass to the
profile of a second portion of said human hematopoietic cell mass
cultured identically but in the absence of said substance or
physical condition.
21. The method of claim 20, wherein said substance is selected from
the group consisting of hematopoietic growth factors, synthetic
agents, hormones and toxins.
22. The method of claim 20, wherein said substance is a monoclonal
antibody specific for a compound endogenously produced by said
hematopoietic cell mass.
23. The method of claim 20, wherein said substance is an antagonist
of a compound endogenously produced by said hematopoietic cell
mass.
24. The method of claim 20, wherein said physical condition is
selected from the group consisting of pressure, temperature,
exposure to light, gravity, and combinations thereof.
25. A functioning in vitro human tissue system, comprising (i) a
chamber containing human hematopoietic cells, including dividing
human hematopoietic stem cells and progenitor cells cultured in a
liquid culture medium, wherein said chamber contains a surface for
the attachment of adherent cells (ii) means for replacing, either
continuously or periodically, said liquid culture medium at a rate
of about 1 ml of medium per ml of culture per about 24 to about 48
hour period, (iii) means for exposing said liquid culture medium to
an oxygen-containing gas, and (iv) means for removing metabolic
products and replenishing depleted nutrients while maintaining said
culture under physiologically acceptable conditions to thereby
maintain a functioning, reconstructed in vitro bone marrow
tissue.
26. The system of claim 25, wherein said chamber comprises two
compartments separated by a membrane which is permeable to oxygen
and carbon dioxide, allows for cell or extracellular matrix
attachment, and is impermeable to water, wherein one of said
compartments contains said liquid culture medium and the other of
said compartments contains said oxygen-containing gas.
27. The system of claim 25, further comprising (v) means for
removing nonadherent cells continuously, periodically, or
intermittently, without disturbing adherent cells.
28. The system of claim 25, further comprising (vi) means for
removing at least a portion of adherent cells in a viable and
functional state.
29. The system of claim 25, further comprising (v) means for
removing nonadherent cells continuously, periodically, or
intermittently, without disturbing adherent cells and (vi) means
for removing at least a portion of adherent cells in a viable and
functional state.
30. The system of claim 25, wherein said human hematopoietic cells
are selected from the group consisting of human periferal blood
mononuclear cells, human bone marrow cells, human fetal liver
cells, human cord blood cells, human spleen cells, and mixtures
thereof.
31. The system of claim 25, wherein said human hematopoietic cells
comprise human stromal stem and/or progenitor cells and/or mature
stromal cells.
32. In a method of bone marrow transplantation, comprising
obtaining a tissue sample from a donor, culturing said tissue
sample, and implanting said tissue sample in a donee, wherein the
improvement is said culturing comprises culturing said tissue,
which comprises a mass of human stem cells, in a liquid culture
medium which is replaced, either continuously or periodically, at a
rate of about 1 ml of medium per ml of culture per about 24 to
about 48 hour period, and removing metabolic products and
replenishing depleted nutrients while maintaining said culture
under physiologically acceptable conditions.
33. The method of claim 32, wherein said tissue sample implanted in
said donee comprises genetically transformed stem and/or progenitor
cells.
34. The method of claim 32, wherein said donee is said donor.
35. The method of claim 32, wherein said donee is not said donor.
Description
[0001] This application is a continuation-in-part of application
Ser. No. 07/740,590, filed Aug. 5, 1991, which is a
continuation-in-part of application Ser. No. 07/737,024, filed Jul.
29, 1991, which is a continuation-in-part of application Ser. No.
07/628,343, filed Dec. 17, 1990, which is a continuation-in-part of
application Ser. No. 07/366,639, filed Jun. 15, 1989, which
disclosures are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to methods and compositions
for the growth of mammalian cells in culture, particularly the
growth of hematopoietic cell cultures. The present invention also
relates to a functioning in vitro human tissue system, which may
serve as a model for hematopoiesis. The present invention further
relates to a method for assaying the effect of a substance and/or
physical condition on a human hematopoietic cell mass or the
hematopoietic process. The present invention also relates to a
method for controlling the lineage development in an in vitro human
tissue system and cultures of cells in which the population of a
particular cell type has been enhanced relative to the total cell
population in the culture or depleted. In addition, the present
invention relates to a method of bone marrow tranplantation, in
which the tissue implanted into the donee has been cultured by the
present method.
[0004] 2. Discussion of the Background
[0005] All of the circulating blood cells in the normal adult,
including erythrocytes, leukocytes, platelets and lymphocytes,
originate as precursor cells within the bone marrow. These cells,
in turn, derive from very immature cells, called progenitors, which
are assayed by their development into contiguous colonies of mature
blood cells in 1-3 week cultures in semisolid media such as
methylcellulose or agar.
[0006] Progenitor cells themselves derive from a class of
progenitor cells called stem cells. Stem cells have the capacity,
upon division, for both self-renewal and differentiation into
progenitors. Thus, dividing stem cells generate both additional
primitive stem cells and somewhat more differentiated progenitor
cells. In addition to the generation of blood cells, stem cells
also may give rise to osteoblasts and osteoclasts, and perhaps
cells of other tissues as well. This document describes methods and
compositions which permit, for the first time, the successful in
vitro culture of human hematopoietic stem cells, which results in
their proliferation and differentiation into progenitor cells and
more mature blood cells of a specific lineage.
[0007] Although there are recent reports of the isolation and
purification of progenitor cells (see, e.g., U.S. Pat. No.
5,061,620 as representative), such methods do not permit the
long-term culture of viable and dividing stem cells.
[0008] In the late 1970s the liquid culture system was developed
for growing hematopoietic bone marrow in vitro. The cultures are of
great potential value both for the analysis of normal and leukemic
hematopoiesis and for the experimental manipulation of bone marrow,
for, e.g., retroviral-mediated gene transfer. These cultures have
allowed a detailed analysis of murine hematopoiesis and have
resulted in a detailed understanding of the murine system. In
addition, it has made possible retroviral gene transfer into
cultured mouse bone marrow cells. This allowed tagging murine
hematopoietic cells proving the existence of the multi-potent stem
cell and of the study of the various genes in the process of
leukemogenesis.
[0009] But while it has been possible to transfer retroviral genes
into cultured mouse bone marrow cells, this has not yet been
possible in cultured human bone marrow cells because, to date,
human long-term bone marrow cultures have been limited both in
their longevity and more importantly in their ability to maintain
stem cell survival and their ability to produce progenitor cells
over time.
[0010] Human liquid bone marrow cultures were initially found to
have a limited hematopoietic potential, producing decreasing
numbers of progenitor cells and mature blood cells, with cell
production ceasing by 6 to 8 weeks. Subsequent modifications of the
original system resulted only in modest improvements. A solution to
this problem is of incalculable value in that it would permit,
e.g., expanding human stem cells and progenitor cells for bone
marrow transplantation and for protection from chemotherapy,
selecting and manipulating such cells, i.e., for gene transfer, and
producing mature human blood cells for transfusion therapy.
[0011] Studies of hematopoiesis and in vitro liquid marrow cultures
have identified fibroblasts and endothelial cells within adhering
layers as central cellular stromal elements. These cells both
provide sites of attachment for developing hematopoietic cells and
can be induced to secrete hematopoietic growth factors which
stimulate progenitor cell proliferation and differentiation. These
hematopoietic growth factors include granulocyte colony stimulating
factor (G-CSF), granulocyte-macrophage colony stimulating factor
(GM-CSF) and interleukin 6 (IL-6).
[0012] Cultures of human bone marrow cells on such adherent layers
in vitro however have been largely disappointing. Unlike related
cultures from other species, such as mouse and tree shrew, human
liquid marrow cultures fail to produce significant numbers of
either nonadherent hematopoietic precursor cells or clonogenic
progenitor cells for over 6 to 8 weeks. And although cultures
lasting 3-5 months have been reported, no culture which stably
produces progenitor cells from stem cells continuously for more
than 4-6 weeks has been reported.
[0013] Moreover, nonadherent and progenitor cell production
typically declined throughout even the short life of these
cultures, so that it is not clear that stem cell survival or
proliferation is supported at all by these cultures. Further, when
studied in isolation, unstimulated bone marrow stromal cells
secrete little if any detectable hematopoietic growth factors
(HGFs).
[0014] The lack of stable progenitor cell and mature blood cell
production in these cultures has led to the belief that they are
unable to support continual stem cell renewal and expansion. It has
therefore been presumed that the cultures either lack a critical
stem cell stimulant(s) and/or contain a novel stem cell
inhibitor(s). However, while explanations for failure to detect
HGFs and uninduced stromal cell cultures have been suggested, the
null hypothesis, which combines the failure to detect HGFs and the
relative failure of human liquid marrow cultures, would be that the
culture systems used in vitro do not provide the full range of
hematopoietic supportive function of adherent bone marrow stromal
cells in vivo.
[0015] Stem cell and progenitor cell expansion for bone marrow
transplantation is a potential application of human long-term bone
marrow cultures. Human autologous and allogeneic bone marrow
transplantation are currently used as therapies for diseases such
as leukemia, lymphoma and other life-threatening disorders. For
these procedures however, a large amount of donor bone marrow must
be removed to insure that there is enough cells for
engraftment.
[0016] A culture providing stem cell and progenitor cell expansion
would reduce the need for large bone marrow donation and would make
possible obtaining a small marrow donation and then expanding the
number of stem cells and progenitor cells in vitro before infusion
into the recipient. Also, it is known that a small number of stem
cells and progenitor cells circulate in the blood stream. If these
stem cells and progenitor cells could be collected by phoresis and
expanded, then it would be possible to obtain the required number
of stem cells and progenitor cells for transplantation from
peripheral blood and eliminate the need for bone marrow
donation.
[0017] Bone marrow transplantation requires that approximately
1.times.10.sup.8 to 2.times.10.sup.8 bone marrow mononuclear cells
per kilogram of patient weight be infused for engraftment. This
requires the bone marrow donation of the same number of cells which
is on the order of 70 ml of marrow for a 70 kg donor. While 70 ml
is a small fraction of the donors marrow, it requires an intensive
donation and significant loss of blood in the donation process. If
stem cells and progenitor cells could be expanded ten-fold, the
donation procedure would be greatly reduced and possibly involve
only collection of stem cells and progenitor cells from peripheral
blood and expansion of these stem cells and progenitor cells.
[0018] Progenitor cell expansion would also be useful as a
supplemental treatment to chemotherapy and is another application
for human long-term bone marrow cultures. The dilemma faced by the
oncologist is that most chemotherapy agents used to destroy cancer
act by killing all cells going through cell division. Bone marrow
is one of the most prolific tissues in the body and is therefore
often the organ that is initially damaged by chemotherapy drugs.
The result is that blood cell production is rapidly destroyed
during chemotherapy treatment and chemotherapy must be terminated
to allow the hematopoietic system to replenish the blood cell
supply before a patient is retreated with chemotherapy. It may take
a month or more for the once quiescent stem cells to raise up the
white blood cell count to acceptable levels to resume chemotherapy
during which case the drop in blood cell count is repeated.
Unfortunately, while blood cells are regenerating between
chemotherapy treatments, the cancer has time to grow and possibly
become more resistant to the chemotherapy drugs due to natural
selection.
[0019] To shorten the time between chemotherapy treatments, large
numbers of progenitor and immature blood cells could be given back
to the patient. This would have the effect of greatly reducing the
time the patient would have low blood cell counts, thereby allowing
more rapid resumption of the chemotherapy treatment. The longer
chemotherapy is given and the shorter the duration between
treatments, the greater the odds of successfully killing the
cancer.
[0020] The hematopoietic cells required for progenitor cell
expansion may come from either bone marrow withdrawal or peripheral
blood collection. Bone marrow harvests would result in collection
of approximately 4.times.10.sup.5 CFU-GM progenitor cells. Phoresis
of 5 liters of peripheral blood would collect approximately
10.sup.5 CFU-GM although this number could be increased to 10.sup.6
CFU-GM by prior treatment of the donor with GM-CSF. Rapid recovery
of a patient would require transfusion of approximately
1.times.10.sup.8 to 5.times.10.sup.8 CFU-GM. Therefore, expansion
of bone marrow or peripheral blood to increase the number of CFU-GM
would be of benefit to chemotherapy administration and cancer
treatment.
[0021] Gene therapy is a rapidly growing field in medicine which is
also of inestimable clinical potential. Gene therapy is, by
definition, the insertion of genes into cells for the purpose of
medicinal therapy. Research in gene therapy has been on-going for
several years in several types of cells in vitro and in animal
studies, and has recently entered the first human clinical trials.
Gene therapy has many potential uses in treating disease and has
been reviewed extensively. See, e.g., Boggs, Int. J. Cell Cloning.
(1990) 8:80-96, Kohn et al, Cancer Invest. (1989) 7 (2):179-192,
Lehn, Bone Marrow Transp. (1990) 5:287-293, and Verma, Scientific
Amer. (1990) pp. 68-84.
[0022] The human hematopoietic system is an ideal choice for gene
therapy in that hematopoietic stem cells are readily accessible for
treatment (bone marrow or peripheral blood harvest), they are
believed to posses unlimited self-renewal capabilities (inferring
lifetime therapy), and upon reinfusion, can expand and repopulate
the marrow. Unfortunately, achieving therapeutic levels of gene
transfer into stem cells has yet to be accomplished in humans.
[0023] Several disorders of the hematopoietic system include
thalassemia, sickle cell anemia, Falconi's anemia, acquired immune
deficiency syndrome (AIDS) and SCIDS (ADA, adenosine deaminase
deficiency). These candidates include both diseases that are
inherited such as hemoglobinopathies and virally caused diseases of
the hematopoietic system such as AIDS.
[0024] A salient problem which remain to be addressed for
successful human gene therapy is the ability to insert the desired
therapeutic gene into the chosen cells in a quantity such that it
will be beneficial to the patient. To date, no method for doing
this is available.
[0025] There is therefore a considerable need for methods and
compositions for the in vitro replication of human stem cells and
for the optimization of human hematopoietic progenitor cell
cultures, particularly in light of the great potential for stem
cell expansion, progenitor cell expansion, and gene therapy offered
by these systems. Unfortunately, to date, attempts to achieve such
results have been disappointing.
[0026] An in vitro system that permitted the controlled production
of specific lineages of blood cells from within a hematopoietic
cell population would have many applications. Controlled production
of red blood cells would permit the in vitro production of red
blood cell units for clinical replacement (transfusion) therapy. As
is well known, red cells transfused are used in the treatment of
anemia following elective surgery, in cases of traumatic blood
loss, and in the supportive care of, e.g., cancer patients.
Similarly, controlled production of platelets would permit the in
vitro production of platelets for platelet transfusion therapy, for
example for cancer patients in whom thrombocytopenia is caused by
chemotherapy. For both red cells and platelets, current volunteer
donor pools are accompanied by the risk of infectious
contamination, and availability of an adequate supply can be
limited. Controlled in vitro production of specified lineage of
mature blood cells circumvent these problems.
[0027] Controlled, selective depletion of a particular lineage of
cells from within a hematopoietic cell population can similarly
confer important advantages. For example, production of stem cells
and myeloid cells while selectively depleting T-cells from a bone
marrow cell population could be very important for the management
of patients with human immunodeficiency virus (HIV) infection.
Since the major reservoir of HIV is the pool of mature T-cells,
selective irradication of the mature T-cells from a hematopoietic
cell mass collected from a patient has considerable potential
therapeutic benefit. If one could selectively remove all the mature
T-cells from within an HIV infected bone marrow cell population
while maintaining viable stem cells, the T-cell depleted bone
marrow sample could then be used to "rescue" the patient following
hematolymphoid ablation and autologous bone marrow transplantation.
Although there are reports of the isolation of progenitor cells
(see, e.g., U.S. Pat. No. 5,061,620 as representative) such
techniques are distinct from and should not be confused with the
selective removal of T-cells from a hematopoietic tissue
culture.
[0028] Another application of T-cell depletion is the prevention of
graft-versus-host disease (GVHD) in allogeneic bone marrow
transplantation. GVHD is a major limiting factor in the success of
allogeneic bone marrow transplantation. Depletion of T-cells from a
stem/progenitor cell population prior to allogeneic transplant
would directly reduce the incidence and severity of GVHD. This
depletion in turn would greatly decrease the morbidity and
mortality of allogeneic, bone marrow transplantation. While there
are currently many techniques available for depleting. T-cells from
bone marrow samples (see e.g. Antin, J. H. et al, Blood, vol. 78,
pp. 2139-2149 (1991)) none of these techniques allow the concurrent
expansion of the hematopoietic progenitor cell population. Thus all
of the previously developed techniques result in a diminution in
the ability of the bone marrow sample to successfully engraft,
thereby resulting in an increased incidence of graft failure. There
is accordingly a considerable need for a method for depleting
T-cells from a human hematopoietic mononuclear cell population,
while maintaining or increasing the hematopoietic progenitor cell
pool within the hematopoietic cell sample.
[0029] In addition, if it were possible to establish a functioning
in vitro human tissue system, one could then utilize such a system
as a model to study the effects of chemical substances and/or
physical conditions on a human hematopoietic cell mass or the
hematopoietic process itself. Thus, by culturing such a system in
the presence of a selected chemical substance and/or physical
condition and comparing the state of the culture (total cell
population, relative abundance of particular cell type,
concentration of cell products in growth medium, etc.) with that of
an identical culture, cultured in the absence of the selected
chemical substance and/or physical condition, it would be possible
to ascertain the effect of the selected chemical substance and/or
physical condition on the hematopoietic cell mass or the
hematopoietic process. In this way, such a functioning in vitro
human tissue system could be utilized in an assay to detect the
effect of a chemical substance and/or physical condition on a human
hematopoietic cell mass or the hematopoietic process itself.
[0030] A further application of selective cell removal is the
purging of malignant cells from bone marrow cultures for autologous
bone marrow transplantation of cancer patients in which the cancer
has metastasized. If it were possible to maintain a viable and
productive human hematopoietic in vitro culture under conditions,
which would lead to the depletion and extinction of malignant
cells, then one could utilize such a culture for an autologous bone
marrow transplant after a bout of chemotherapy, without the
consequence of reintroducing metastasized malignant cells to the
patient via the bone marrow transplant.
[0031] Thus, there remains a need for a functioning in vitro human
hematopoietic tissue system and methods and conditions for
maintaining such a system.
SUMMARY OF THE INVENTION
[0032] Accordingly, it is an object of this invention to provide
novel methods, including culture media conditions, for the in vitro
replication of human stem cells.
[0033] It is another object of this invention to provide novel
methods, including culture media conditions, for the optimization
of human hematopoietic progenitor cell cultures.
[0034] It is another object of the present invention to provide a
novel, functioning, in vitro hematopoietic tissue system which may
serve as a model of hematopoiesis.
[0035] It is another object of the present invention to provide a
novel, functioning, in vitro hematopoietic tissue system which is
substantially free of T-cells and B-cells.
[0036] It is another object of the present invention to provide a
novel, functioning, in vitro hematopoietic tissue system in which
at least a portion of the stem cells present have been genetically
transformed.
[0037] It is another object of this invention to provide a novel,
functioning, in vitro bone marrow tissue system in which the
lineages of blood cells, including stem cells, produced can be
controlled.
[0038] It is another object of this invention to provide novel
methods, including culture media conditions, for the optimization
of human hematopoietic progenitor cell cultures and to control the
lineage composition of the mature cells produced.
[0039] It is another object of the present invention to provide
novel methods, including culture media conditions, for the
optimization of human hematopoietic progenitor cell cultures and to
control the linage composition of the mature cells produced, in
which at least a portion of the mature cells are derived from stem
cells which have been genetically transformed.
[0040] It is another object of this invention to provide novel
methods, including culture media conditions, for the selective
enhanced production of red blood cells.
[0041] It is another object of this invention to provide novel
methods, including culture media conditions, for the depletion of
T-cells and B-cells from a human hematopoietic cell population.
[0042] It is another object of this invention to provide novel
methods, including culture media conditions, for removing malignant
cells from a human hematopoietic cell population.
[0043] It is another object of this invention to provide novel
methods, including culture media conditions, for assaying the
affect of a substance or substances on a human replicating
hematopoietic cell population.
[0044] It is another object of the present invention to provide
novel methods, including culture media conditions, for assaying the
effect of a physical condition or conditions on a human replicating
hematopoietic cell population.
[0045] It is another object of the present invention to provide
novel methods, including culture media conditions, for assaying the
effect of genetic transformation of stem cells on a human
replicating hematopoietic cell population.
[0046] It is another object of the present invention to provide
novel methods for performing bone marrow transplantation in which
the bone marrow tissue implanted in a patient is obtained according
to the present method.
[0047] It is another object of the present invention to provide
novel methods for performing bone marrow transplantation in which
the bone marrow tissue implanted in a patient has been depleted of
T-cells and B-cells.
[0048] It is another object of the present invention to provide
novel methods for performing bone marrow transplantation in which
the bone marrow tissue implanted in a patient has been depleted of
malignant cells.
[0049] It is another object of the present invention to provide
novel methods for performing bone marrow transplantation in which
the bone marrow tissue implanted in a patient has been enriched in
the population of a particular cell type as compared to the total
cell population.
[0050] It is another object of the present invention to provide
novel methods for performing bone marrow transplantation in which
the bone marrow tissue implanted in a patient comprises stem cells
which have been genetically transformed.
[0051] The present invention is based on the inventors' discovery
of novel methods, including culture media conditions, which provide
for in vitro human stem cell division and/or the optimization of
human hematopoietic progenitor cell cultures. These methods rely on
culturing human stem cells and/or human hematopoietic progenitor
cells in a liquid culture medium which is replaced, preferably
perfused, either continuously, periodically, or intermittently, at
a rate of 1 milliliter (ml) of medium per ml of culture per about
24 to about 48 hour period, and removing metabolic products and
replenishing depleted nutrients while maintaining the culture under
physiologically acceptable conditions. In a particularly preferred
embodiment of the present invention, the above medium replacement
rate is used in conjunction with the addition of hematopoietic
growth factors to the rapidly exchanged culture medium.
[0052] The inventors have discovered that the increased medium
exchange rate used in accordance with the present invention, with
the optional addition of hematopoietic growth factors to the
rapidly exchanged culture medium, surprisingly (1) supports
cultures in which human stem cells proliferate over extended
periods of time of at least 5 months, (2) supports cultures in
which human hematopoietic progenitor cells are produced by division
and differentiation of human stem cells through extended culture
periods of at least 5 months, (3) stimulates the increased
metabolism of and growth factor, including GM-CSF, secretion from
human stromal cells, including human bone marrow stromal cells, (4)
provides for the depletion of T-cells and B-cells from a human
hematopoietic mononuclear cell population, (5) provides a method
for assaying the affect of a substance or substances or physical
conditions on a human hematopoietic cell population, (6) provides
for the depletion of malignant cells from a human hematopoietic
cell population, and (7) supports cultures in which human stem
cells continue to divide over long periods of time and, thus, may
be genetically transformed with a suitable vector such as a
retrovirus. The present invention provides, for the first time,
human stem cell survival and proliferation in culture. In addition,
the present invention provides a functioning human in vitro tissue
system which may serve as a model for a human hematopoietic cell
mass or the process of hematopoiesis.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0053] The advantages of the present invention may be observed
whenever the present invention is applied to any standard system
for liquid human hematopoietic culture. By the use of the rapid
medium exchange rates used in accordance with the present
invention, with the optional addition of supplementary
hematopoietic growth factors to the culture, the inventors have
surprisingly discovered that one is able to make standard systems
for liquid human hematopoietic cultures, which comprise cultures
performed in the presence or absence of animal sera or plasmas,
including horse, calf, fetal calf, or human serum, perform in a
qualitatively superior manner. Human liquid hematopoietic cultures
which may be used in accordance with the invention can be performed
at cell densities of from 10.sup.4 to 5.times.10.sup.8 cells per ml
of culture, using standard known medium components such as, for
example, IMDM, MEM, DMEM, RPMI 1640, Alpha Medium or McCoy's
Medium, which can use combinations of serum albumin, cholesterol
and/or insulin, transferrin, lecithin, selenium and inorganic
salts. As known, these cultures may be supplemented with
corticosteroids, such as hydrocortisone at a concentration of
10.sup.-4 to 10.sup.-7 M, or other corticbsteroids at equal potent
dose such as cortisone, dexamethasone or Solu-Medrols.RTM.
(Upjohn).
[0054] These cultures are typically carried out at a pH which is
roughly physiologic, i.e. 6.9 to 7.6. The medium is kept at an
oxygen concentration that corresponds to an oxygen-containing
atmosphere which contains from 1 to 20 vol. percent oxygen,
preferably 3 to 12 vol. percent oxygen. The preferred range of
O.sub.2 concentration refers to the concentration of O.sub.2 near
the cells, not necessarily at the point of O.sub.2 introduction
which may be at the medium surface or through a membrane. Using
these standard culture techniques, the cell mass used may be
enriched, by any desired amount, such as by up to 10.sup.3 fold or
more, either for stem cell content or for hematopoietic progenitor
cell content. Different known methods may be used to achieve this
enrichment, corresponding either to a negative selection method or
a positive selection method. For example, in accordance with the
negative selection method, mature cells are removed using
immunological techniques, e.g., labelling non-progenitor, non-stem
cells with a panel of mouse anti-human monoclonal antibodies, then
removing the mouse antibody-coated cells by adherence to
rabbit-anti-mouse Ig-coated plastic dishes. See e.g., Emerson et
al, J. Clin. Invest. (1985) 76:1286-1290. Via such procedures, stem
cells and progenitor cells may be concentrated to any degree
desired.
[0055] The present invention relies on a fundamental alteration of
the conditions of liquid human bone marrow cultures under any of
the above conditions; rapid replacement of the nutrient medium.
Standard culture schedules call for medium and serum to be
exchanged weekly, either as a single exchange performed weekly or a
one-half medium and serum exchange performed twice weekly. In
accordance with the present invention, the nutrient medium of the
culture is replaced, preferably perfused, either continuously or
periodically, at a rate of about 1 ml per ml of culture per about
24 to about 48 hour period, for cells cultured at a density of from
2.times.10.sup.6 to 1.times.10.sup.7 cells per ml. For cell
densities of from 1.times.10.sup.4 to 2.times.10.sup.6 cells per ml
the same medium exchange rate may be used. Thus, for cell densities
of about 10.sup.7 cells per ml, the present medium replacement rate
may be expressed as 1 ml of medium per 10.sup.7 cells per about 24
to about 48 hour period. For cell densities higher than 10.sup.7
cells per ml, the medium exchange rate may be increased
proportionality to achieve a constant medium and serum flux per
cell per unit time. Replacement of the nutrient medium in
accordance with the invention may be carried out in any manner
which will achieve the result of replacing the medium, e.g., by
removing an aliquot of spent culture medium and replacing it with a
fresh aliquot. The flow of the aliquot being added may be by
gravity, by pump, or by any other suitable means, such as syringe
or pipette. The flow may be in any direction or multiplicity of
directions, depending upon the configuration and packing of the
culture. Preferably, the new medium is added to the culture in a
manner such that it contacts the cell mass. Most preferably, it is
added to the culture in a manner mimicking in vivo perfusion, i.e.,
it is perfused through at least part of the cell mass and up to the
whole cell mass.
[0056] Another, optional but important, embodiment of the present
invention, resides in the addition of hematopoietic growth factors
to the rapidly exchanged cultures. In a particularly preferred
aspect of this embodiment, the cytokines IL-3 and GM-CSF are both
added, together, to the medium at a rate of from 0.1 to 100
ng/ml/day, preferably about 0.5 to 10 ng/ml/day, most preferably 1
to 2 ng/ml/day. Epo may be added to the nutrient medium in an
amount of from 0.001 to 10 U/ml/day, preferably 0.05 to 0.15
U/ml/day. Mast cell growth factor (MCF, c-kit ligand, Steel
factor), may be added to the medium in an amount of from 1 to 100
ng/ml/day, preferably 10 to 50 ng/ml/day. IL-1 (.alpha. or .beta.)
may also be added in an amount of from 10 to 100 units/ml per 3 to
5 day period. Additionally, IL-6, G-CSF, basic fibroblast growth
factor, IL-7, IL-8, IL-9, IL-10, IL-11, PDGF, or EGF may be added,
at a rate of from 1 to 100 ng/ml/day.
[0057] The metabolic product level in the medium is normally
maintained within a particular range. Glucose concentration is
usually maintained in the range of about 5 to 20 mM. Lactate
concentration is usually maintained below 35 mM. Glutamine
concentration is generally maintained in the range of from about 1
to 3 mM. Ammonium concentration is usually maintained below about
2.4 mM. These concentrations can be monitored by either periodic
off line or on line continuous measurements using known methods.
See, e.g., Caldwell et al, J. Cell Physiol. (1991) 147:344-353. The
cells which may be cultured in accordance with the present
invention may be human peripheral blood mononuclear cells, human
bone marrow cells, human fetal liver cells, human cord blood cells
and/or human spleen cells. Each of these cell masses contains human
stem cells and human hematopoietic progenitor cells.
[0058] In a preferred embodiment of the invention, the cell culture
may be enriched to augment the human stem cell content of the cell
mass. Such enrichment may achieved as described above, and, when
used in accordance with the invention, provides the first useful
means for genetic therapy via gene transfer into human bone marrow
stem cells. In this embodiment, a packing cell line infected with a
retrovirus, or a supernatant obtained from such a packaging cell
line culture, is added to human stem cells cultured in accordance
with the invention to obtain transformed human bone marrow stem
cells. Such genetic transformation of human stem cells may be
carried out as described in U.S. patent application Ser. No.
07/740,590, which is incorporated herein by reference. The present
invention provides increased levels of stem cell and human
hematopoietic progenitor cell replication, whereas, by contrast,
prior cultures provided only for human hematopoietic progenitor
cell replication at a decreasing rate (i.e., decaying cultures).
The present culture system provides, for the first time, expansion
of cells in culture, which is required for retroviral infection of
cells. Earlier systems in which retroviral infection was carried
out on decaying cultures provided no infection of earlier cells.
The present invention, particularly when it is practiced together
with an enriched stem cell pool, and even more particularly when it
is practiced still further with the use of hematopoietic growth
factors, provides a very effective means for obtaining stem cell
infection in vitro.
[0059] In accordance with the present invention one obtains
cultures in which human hematopoietic progenitor cells are produced
by division and differentiation from human stem cells throughout a
culture period of at least five months. That is, one obtains a
culture which supports stem cell survival and proliferation in
culture.
[0060] Data obtained by the inventors indicates that medium
perfusion rate is a very significant variable in determining the
behavior of in vitro human bone marrow cultures. This data shows
that when the medium exchange rate is increased from the
traditional once per week Dexter rate to a daily medium exchange
rate of 7 volumes per week, a significant effect on in vitro
hematopoiesis is obtained. In experiments carried out by the
inventors, all cultures displayed a significant loss of cells
during the first 3 to 4 weeks. Following this decay, the cultures
stabilized and the effect of a medium perfusion rate became more
pronounced.
[0061] A 3.5 per week medium exchange rate led to the most prolific
cultures in the absence of added growth factors and also to
cultures of greatest longevity in terms of progenitor cell
production. Of particular note, during weeks 4 to 10, the biweekly
number of nonadherent cells produced was actually stable or
increasing.
[0062] Over the entire course of the cultures, the cumulative
number of cells produced after week 3.5 was almost three-fold
greater than that which is produced under the traditional Dexter
culture protocol. Further, stable production of progenitor cells is
maintained until week 18.
[0063] Bone marrow stomal cells may or may not be present in the
cultures of the invention. In typical cultures, stromal cells are
present in the cell culture in an amount of approximately 10.sup.-3
to 10.sup.-1 (stromal cells/total cells).
[0064] In another aspect of the invention, the inventors discovered
that the cultures of the invention surprisingly provide increased
metabolism and GM-CSF and IL-6 secretion from human bone marrow
stromal cells. Whereas no GM-CSF is detected in human bone marrow
stromal cells supernatant, rapid medium exchange in accordance with
the invention stimulates human bone marrow stromal cells to secrete
300 femtograms/ml/day to 200 picograms/ml/day of GM-CSF. Secretion
of IL-6 by human bone marrow stromal cells is also increased by
rapid medium exchange in accordance with the invention from 1 to 2
ng/ml/day to 2 to 4 ng/ml/day. This increase is observed both when
only the rapid medium exchange rate of the invention is used, and
when the rapid exchange rate together with the addition of
hematopoietic growth factors is used. On the basis of data obtained
by the inventors, the effect of the rapid medium exchange rates of
the invention on human stromal cell production of cytokines should
be observed with human stromal cells in any complex tissue culture
system.
[0065] Illustratively, the medium used in accordance with the
invention may comprise three basic components. The first component
is a media component comprised of IMDM, MEM, DMEM, RPMI 1640, Alpha
Medium or McCoy's Medium, or an equivalent known culture medium
component. The second is a serum component which comprises at least
horse serum or human serum and may optionally further comprise
fetal calf serum, newborn calf serum, and/or calf serum. The third
component is a corticosteroid, such as hydrocortisone, cortisone,
dexamethasone, Solu-Medrol.RTM. (Upjohn), or a combination of
these, preferably hydrocortisone. The serum component can be
replaced in whole or in part with any standard serum replacement
mixture.
[0066] The compositional make up of various media which can be used
are set forth below.
1 Dulbecco's.sup.1 Modified Eagle Media (D-MEM) 320-1885 380-2320
430-1600 320-1965 380-2430 430-2100 430-2800 1X Liquid 1X Liquid
Powder 1X Liquid 1X Liquid Powder Powder COMPONENT mg/L mg/L mg/L
mg/L mg/L mg/L mg/L INORGANIC SALTS: CaCl.sub.2 (anhyd.) 200.00
200.00 200.00 200.00 200.00 200.00 200.00
Fe(NO.sub.3).sub.3.9H.sub.2O 0.10 0.10 0.10 0.10 0.10 0.10 0.10 KCl
400.00 400.00 400.00 400.00 400.00 400.00 400.00 MgSO.sub.4
(anhyd.) -- -- 97.67 -- -- 97.67 97.67 MgSO.sub.4.7H.sub.2O 200.00
200.00 -- 200.00 200.00 -- -- NaCl 6400.00 4750.00 6400.00 6400.00
4750.00 6400.00 6400.00 NaHCO.sub.3 3700.00 3700.00 -- 3700.00
3700.00 -- -- NaH.sub.2PO.sub.4.H.sub.2O.sup.a 125.00 125.00 125.00
125.00 125.00 125.00 125.00 OTHER COMPONENTS: D-Glucose 1000.00
1000.00 1000.00 4500.00 4500.00 4500.00 4500.00 Phenol red 15.00
15.00 15.00 15.00 15.00 15.00 15.00 HEPES -- 5958.00 -- -- 5958.00
-- -- Sodium pyruvate 110.00 110.00 110.00 -- -- -- 110.00 AMINO
ACIDS: L-Arginine-HCl 84.00 84.00 84.00 84.00 84.00 84.00 84.00
L-Cystine 48.00 48.00 -- 48.00 48.00 -- -- L-Cytine-2HCl -- --
62.57 -- -- 62.57 62.57 L-Glutamine 584.00 584.00 584.00 584.00
584.00 584.00 584.00 Glycine 30.00 30.00 30.00 30.00 30.00 30.00
30.00 L-Histidine-HCl.H.sub.2O 42.00 42.00 42.00 42.00 42.00 42.00
42.00 L-Isoleucine 105.00 105.00 105.00 105.00 105.00 105.00 105.00
L-Leucine 105.00 105.00 105.00 105.00 105.00 105.00 105.00
L-Lysine-HCl 146.00 146.00 146.00 146.00 146.00 146.00 146.00
L-Methionine 30.00 30.00 30.00 30.00 30.00 30.00 30.00
L-Phenylalanine 66.00 66.00 66.00 66.00 66.00 66.00 66.00 L-Serine
42.00 42.00 42.00 42.00 42.00 42.00 42.00 L-Threonine 95.00 95.00
95.00 95.00 95.00 95.00 95.00 L-Tryptophan 16.00 16.00 16.00 16.00
16.00 16.00 16.00 L-Tyrosine 72.00 72.00 -- 72.00 72.00 -- --
L-Tyrosine-2Na.2H.sub.20 -- -- 103.79 -- -- 103.79 103.79 L-Valine
94.00 94.00 94.00 94.00 94.00 94.00 94.00 VITAMINS: D-Ca
pantothenate 4.00 4.00 4.00 4.00 4.00 4.00 4.00 Choline chloride
4.00 4.00 4.00 4.00 4.00 4.00 4.00 Folic acid 4.00 4.00 4.00 4.00
4.00 4.00 4.00 i-Inositol 7.20 7.20 7.20 7.20 7.20 7.20 7.20
Niacinamide 4.00 4.00 4.00 4.00 4.00 4.00 4.00 Pyridoxal-HCl 4.00
4.00 4.00 4.00 4.00 4.00 4.00 Riboflavin 0.40 0.40 0.40 0.40 0.40
0.40 0.40 Thiamine-HCl 4.00 4.00 4.00 4.00 4.00 4.00 4.00 430-3000
320-1968 320-1970 320-1995 430-3700 320-1968 430-3000 Powder 1X
Liquid 1X Liquid 1X Liquid Powder 1X Liquid Powder COMPONENT mg/L
mg/L mg/L mg/L mg/L mg/L mg/L INORGANIC SALTS: CaCl.sub.2 (anhyd.)
200.00 200.00 200.00 200.00 200.00 200.00 200.00
Fe(NO.sub.3).sub.3.9H.su- b.2O 0.10 0.10 0.10 0.10 0.10 0.10 0.10
KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00 MgSO.sub.4
(anhyd.) 97.67 -- -- -- 97.67 -- 97.67 MgSO.sub.4.7H.sub.2O --
200.00 200.00 200.00 -- 200.00 -- NaCl 6400.00 6400.00 6400.00
6400.00 4750.00 6400.00 6400.00 NaHCO.sub.3 -- 3700.00 3700.00
3700.00 -- 3700.00 -- NaH.sub.2PO.sub.4.H.sub.2O.sup.a 125.00
125.00 125.00 125.00 125.00 125.00 125.00 OTHER COMPONENTS:
D-Glucose 4500.00 4500.00 4500.00 4500.00 4500.00 4500.00 -- Phenol
red -- 15.00 15.00 15.00 15.00 -- HEPES -- -- -- -- 5958.00 -- --
Sodium pyruvate -- -- -- 110.00 -- -- -- AMINO ACIDS:
L-Arginine-HCl 84.00 84.00 84.00 84.00 84.00 84.00 84.00 L-Cystine
-- 48.00 48.00 48.00 -- 48.00 -- L-Cytine-2HCl -- -- -- 62.57 --
62.57 L-Glutamine 584.00 -- -- 584.00 584.00 584.00 -- Glycine
30.00 30.00 30.00 30.00 30.00 30.00 30.00 L-Histidine-HCl.H.sub.2O
42.00 42.00 42.00 42.00 42.00 42.00 42.00 L-Isoleucine 105.00
105.00 105.00 105.00 105.00 105.00 105.00 L-Leucine 105.00 105.00
105.00 105.00 105.00 105.00 L-Lysine-HCl 146.00 146.00 146.00
146.00 146.00 146.00 146.00 L-Methionine 30.00 30.00 -- 30.00 30.00
30.00 30.00 L-Phenylalanine 66.00 66.00 66.00 66.00 66.00 66.00
66.00 L-Serine 42.00 42.00 42.00 42.00 42.00 42.00 42.00
L-Threonine 95.00 95.00 95.00 95.00 95.00 95.00 95.00 L-Tryptophan
16.00 16.00 16.00 16.00 16.00 16.00 16.00 L-Tyrosine -- 72.00 72.00
72.00 -- 72.00 -- L-Tyrosine-2Na.2H.sub.20 103.79 -- -- -- 103.79
-- 103.79 L-Valine 94.00 94.00 94.00 94.00 94.00 94.00 94.00
VITAMINS: D-Ca pantothenate 4.00 4.00 4.00 4.00 4.00 4.00 4.00
Choline chloride 4.00 4.00 4.00 4.00 4.00 4.00 4.00 Folic acid 4.00
4.00 4.00 4.00 4.00 4.00 4.00 i-Inositol 7.20 7.20 7.20 7.20 7.20
-- 7.20 Niacinamide 4.00 4.00 4.00 4.00 4.00 4.00 4.00
Pyridoxal-HCl 4.00 4.00 4.00 4.00 4.00 4.00 4.00 Riboflavin 0.40
0.40 0.40 0.40 0.40 0.40 0.40 Thiamine-HCl 4.00 4.00 4.00 4.00 4.00
4.00 4.00 .sup.1Dulbecco, R. and Freeman, G. (1959) Virology 8,
396. Smith, J. D., Freeman, G., Vogt, M., and Dulbecco, R. (1960)
Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:
2, 93. .sup.aValues shown are in conformance with the Tissue
Culture Standards Committee, In Vitro (1970) 9:6.
[0067]
2 DMEM/F12 320-1330 430-2400 320-1320 430-2900 1X Liquid Powder 1X
Liquid Powder COMPONENT mg/L mg/L mg/L mg/L INORGANIC SALTS:
CaCl.sub.2 (anhyd.) 116.60 116.60 116.60 116.60
CuSO.sub.4.5H.sub.2O 0.0013 0.0013 0.0013 0.0013
Fe(NO.sub.3).sub.3.9H.sub.2O 0.05 0.05 0.05 0.05
FeSO.sub.4.7H.sub.2O 0.417 0.417 0.417 0.417 KCl 311.80 311.80
311.80 311.80 MgCl.sub.2 -- 28.64 28.64 28.64 MgCl.sub.2.6H.sub.2O
61.00 -- -- -- MgSO.sub.4 -- 48.84 48.84 48.84 MgSO.sub.4.7H.sub.2O
100.00 -- -- -- NaCl 6999.50 6999.50 6999.50 6999.50 NaHCO.sub.3
1200.00 -- 2438.00 -- NaH.sub.2PO.sub.4.H.sub.2O 62.50 62.50 62.50
62.50 Na.sub.2HPO.sub.4 -- 71.02 71.02 71.02
Na.sub.2HPO.sub.4.7H.sub.2O 134.00 -- -- -- ZnSO.sub.4.7H.sub.2O
0.432 0.432 0.432 0.432 OTHER COMPONENTS: D-Glucose 3151.00 3151.00
3151.00 3151.00 HEPES 3574.50 3574.50 -- -- Na hypoxanthine 2.39
2.39 2.39 2.39 Linoleic acid 0.042 0.042 0.042 0.042 Lipoic acid
0.105 0.105 0.105 0.105 Phenol red 8.10 8.10 8.10 8.10
Putreacine-2HCl 0.081 0.081 0.081 0.081 Sodium pyruvate 55.00 55.00
55.00 55.00 AMINO ACIDS: L-Alanine 4.45 4.45 4.45 4.45 L-Arginine
HCl 147.50 147.50 147.50 147.50 L-Asparagine-H.sub.2O 7.50 7.50
7.50 7.50 L-Aspartic acid 6.65 6.65 6.65 6.65
L-Cystine-HCl.H.sub.2O 17.56 17.56 17.56 17.56 L-Cystine-2HCl 31.29
31.29 31.29 31.29 L-Glutamic acid 7.35 7.35 7.35 7.35 L-Glutamine
365.00 365.00 365.00 365.00 Glycine 18.75 18.75 18.75 18.75
L-Histidine-HCl.H.sub.2O 31.48 31.48 31.48 31.48 L-Isoleucine 54.47
54.47 54.47 54.47 L-Leucine 59.05 59.05 59.05 59.05 L-Lysine-HCl
91.25 91.25 91.25 91.25 L-Methionine 17.24 17.24 17.24 17.24
L-Phenylalanine 35.48 35.48 35.48 35.48 L-Proline 17.25 17.25 17.25
17.25 L-Serine 26.25 26.25 26.25 26.25 L-Threonine 53.45 53.45
53.45 53.45 L-Tryptophan 9.02 9.02 9.02 9.02
L-Tyrosine-2Na.2H.sub.2O 55.79 55.79 55.79 55.79 L-Valine 52.85
52.85 52.85 52.85 VITAMINS: Biotin 0.0035 0.0035 0.0035 0.0035 D-Ca
pantothenate 2.24 2.24 2.24 2.24 Choline chloride 8.98 8.98 8.98
8.98 Folic acid 2.65 2.65 2.65 2.65 i-Inositol 12.60 12.60 12.60
12.60 Niacinamide 2.02 2.02 2.02 2.02 Pyridoxal-HCl 2.00 2.00 2.00
2.00 Pyridoxine-HCl 0.031 0.031 0.031 0.031 Riboflavin 0.219 0.219
0.219 0.219 Thiamine-HCl 2.17 2.17 2.17 2.17 Thymidine 0.365 0.365
0.365 0.365 Vitamin B.sub.12 0.68 0.68 0.68 0.68
[0068]
3 Iscove's Modified Dulbecco's Media (IMDM).sup.1,2,3 380-2440
430-2200 1X Liquid Powder COMPONENT mg/L mg/L INORGANIC SALTS:
CaCl.sub.2 (anhyd.) 165.00 165.00 KCl 330.00 330.00 KNO.sub.3 0.076
0.076 MgSO.sub.4 (anhyd.) 97.67 97.67 NaCl 4505.00 4505.00
NaHCO.sub.3 3024.00 -- NaH.sub.2PO.sub.4.multidot.H.sub.2O.sup.a
125.00 125.00 Na.sub.2SeO.sub.35H.sub.2O 0.0173 0.0173 OTHER
COMPONENTS: D-Glucose 4500.00 4500.00 Phenol red 15.00 15.00 HEPES
5958.00 5958.00 Sodium pyruvate 110.00 110.00 AMINO ACIDS:
L-Alanine 25.00 25.00 L-Asparagine-H.sub.2O 28.40 28.40
L-Arginine-HCl 84.00 84.00 L-Aspartic acid 30.00 30.00
L-Cystine-2HCl 91.24 91.24 L-Glutamic acid 75.00 75.00 L-Glutamine
584.00 584.00 Glycine 30.00 30.00 L-Histidine-HCl.multidot.H.sub.2O
42.00 42.00 L-Isoleucine 105.00 105.00 L-Leucine 105.00 105.00
L-Lysine-HCl 146.00 146.00 L-Methionine 30.00 30.00 L-Phenylalanine
66.00 66.00 L-Proline 40.00 40.00 L-Serine 42.00 42.00 L-Threonine
95.00 95.00 L-Tryptophan 16.00 16.00
L-Tyrosine-2Na.multidot.2H.sub.2O 103.79 103.79 L-Valine 94.00
94.00 VITAMINS: Biotin 0.013 0.013 D-Ca pantothenate 4.00 4.00
Choline chloride 4.00 4.00 Folic acid 4.00 4.00 i-Inositol 7.20
7.20 Niacinamide 4.00 4.00 Pyridoxal-HCl 4.00 4.00 Riboflavia 0.40
0.40 Thiamine-HCl 4.00 4.00 Vitamin B.sub.12 0.013 0.013
.sup.1Dulbecco, R. and Freeman, G. (1959) Virology 8, 396. Smith,
J. D., Freeman, G., Vogt, M., and Dulbecco, R. (1960) Virology 12,
185, Tissue Culture Standards Committee, In Vitro 6:2, 93.
.sup.2Iscove, N. N. and Melchers, F., J. Experimental Medicine 147,
923. .sup.aValues shown are in conformance with Tissue Culture
Standards Committee, In Vitro (1970) 9: 6. .sup.3Iscove, N. N.,
personal communication.
[0069]
4 McCoy's 5A Media (modified).sup.1,2,3 320-6600 380-2330 430-1500
320-6608 320-6601.sup.4 320-6610 320-6620 320-6630 1X Liquid 1X
Liquid Powder 1X Liquid 1X Liquid 1X Liquid 1X Liquid 1X Liquid
COMPONENT mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L INORGANIC SALTS:
CaCl.sub.2 (anhyd.) 100.00 100.00 100.00 -- 140.00 100.00 100.00
100.00 KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00
KH.sub.2PO.sub.4 -- -- -- -- 60.00 -- -- -- MgCl.sub.2.6H.sub.2O --
-- -- -- 100.00 -- -- -- MgSO.sub.4 (anhyd.) -- -- 97.67 -- -- --
-- -- MgSO.sub.4.7H.sub.2O 200.00 200.00 -- 200.00 100.00 200.00
200.00 200.00 NaCl 6460.00 5100.00 6460.00 6460.00 8000.00 6460.00
6460.00 6460.00 NaHCO.sub.3 2200.00 2200.00 -- 2200.00 350.00
2200.00 2200.00 2200.00 NaH.sub.2PO.sub.4.H.sub.2O 580.00 580.00
580.00 1400.00 -- 580.00 580.00 580.00 Na.sub.2HPO.sub.4.7H.sub.2O
-- -- -- -- 90.00 -- -- -- OTHER COMPONENTS: Bacto-peptone 600.00
600.00 600.00 600.00 600.00 600.00 600.00 600.00 Fetal Bovine Serum
-- -- -- -- -- c c c D-Glucose 3000.00 3000.00 3000.00 3000.00
1000.00 3000.00 3000.00 3000.00 Glutathione (reduced) 0.50 0.50
0.50 0.50 0.50 0.50 0.50 0.50 HEPES -- 5958.00 -- -- -- -- -- --
Phenol red 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 AMINO
ACIDS: L-Alanine 13.90 13.90 13.90 13.90 13.90 13.90 13.90 13.90
L-Arginine-HCl 42.10 42.10 42.10 42.10 42.10 42.10 42.10 42.10
L-Asparagine.sup.a 45.00 45.00 45.00 45.00 45.00 45.00 45.00 45.00
L-Aspartic acid 19.97 19.97 19.97 19.97 19.97 19.97 19.97 19.97
L-Cysteine.sup.b 31.50 31.50 31.50 31.50 31.50 31.50 31.50 31.50
L-Glutamic acid 22.10 22.10 22.10 22.10 22.10 22.10 22.10 22.10
L-Glutamine 219.20 219.20 219.20 219.20 219.20 219.20 219.20 219.20
Glycine 7.50 7.50 7.50 7.50 7.50 7.50 7.50 7.50
L-Histidine-HCl.H.sub.2O 20.96 20.96 20.96 20.96 20.96 20.96 20.96
20.96 L-Hydroxyproline 19.70 19.70 19.70 19.70 19.70 19.70 19.70
19.70 L-Isoleucine 39.36 39.36 39.36 39.36 39.36 39.36 39.36 39.36
L-Leucine 39.36 39.36 39.36 39.36 39.36 39.36 39.36 39.36
L-Lysine-HCl 36.50 36.50 36.50 36.50 36.50 36.50 36.50 36.50
L-Methionine 14.90 14.90 14.90 14.90 14.90 14.90 14.90 14.90
L-Phenylalanine 16.50 16.50 16.50 16.50 16.50 16.50 16.50 16.50
L-Proline 17.30 17.30 17.30 17.30 17.30 17.30 17.30 17.30 L-Serine
26.30 26.30 26.30 26.30 26.30 26.30 26.30 26.30 L-Threonine 17.90
17.90 17.90 17.90 17.90 17.90 17.90 17.90 L-Tryptophan 3.10 3.10
3.10 3.10 3.10 3.10 3.10 3.10 L-Tyrosine 18.10 18.10 -- 18.10 18.10
18.10 18.10 18.10 L-Tyrosine-2Na.2H.sub.2O -- -- 26.10 -- -- -- --
-- L-Valine 17.60 17.60 17.60 17.60 17.60 17.60 17.60 17.60
VITAMINS: Ascorbic acid 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
Biotin 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 Choline chloride
5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 D-Ca pantothenate 0.20 0.20
0.20 0.20 0.20 0.20 0.20 0.20 Folic acid 10.00 10.00 10.00 10.00
10.00 10.00 10.00 10.00 i-Inositol 36.00 36.00 36.00 36.00 36.00
36.00 36.00 36.00 Niacinamide 0.50 0.50 0.50 0.50 0.50 0.50 0.50
0.50 Nicotinic acid 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
Para-aminobenzoic acid 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
Pyridoxal-HCl 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
Pyridoxine-HCl 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 Riboflavin
0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 Thiamine.HCl 0.20 0.20 0.20
0.20 0.20 0.20 0.20 0.20 Vitamin B.sub.12 2.00 2.00 2.00 2.00 2.00
2.00 2.00 2.00 .sup.1McCoy, T. A., Maxwell, M., and Kruse, P. F.
(1959) Proc. Soc. Exper. Biol. Med. 100, 115. .sup.2Hsu, T. C. and
Kellogg, D. S., Jr. (1960) J. Nat. Cancer Inst. 25, 221.
.sup.3Iwakata, S. and Grace, J. T., Jr. (1964) N. Y. J. Med. 64:
18, 2279. .sup.4McCoy's 5A Medium formulated with Hanks' and
Suspension Salts is a GIBCO modification and is not cited in
references 1-3. .sup.aHCl form listed by the Tissue Culture
Standards Committee, In Vitro (1974) 9: 6. .sup.bMonohydrate form
listed by the Tissue Culture Standards Committee, In Vitro (1974)
9: 6. c Fetal Bovine Serum Supplementation: Cat. No. FBS 320-6610
10% v/v 320-6620 20% v/v 320-6630 30% v/v
[0070]
5 Minimum Essential Media (MEM).sup.1 320-2561.sup.2 410-2000.sup.2
320-2571.sup.2 410-1900.sup.2 320-2570 320-1090 380-2360 1X Liquid
Powder 1X Liquid Powder 1X Liquid 1X Liquid 1X Liquid COMPONENT
mg/L mg/L mg/L mg/L mg/L mg/L mg/L INORGANIC SALTS: CaCl.sub.2
(anhyd.) 200.00 200.00 200.00 200.00 200.00 200.00 200.00 KCl
400.00 400.00 400.00 400.00 400.00 400.00 400.00 MgSO.sub.4
(anhyd.) -- 97.67 -- 97.67 -- -- -- MgSO.sub.4.7H.sub.2O 200.00 --
200.00 -- 200.00 200.00 200.00 NaCl 6800.00 6800.00 6800.00 6800.00
6800.00 6800.00 6350.00 NaHCO.sub.3 2200.00 -- 2200.00 -- 2200.00
2200.00 2200.00 NaH.sub.2PO.sub.4.H.sub.2O.sup.a 140.00 140.00
140.00 140.00 140.00 140.00 140.00 OTHER COMPONENTS: D-Glucose
1000.00 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00 HEPES -- --
-- -- -- -- 5958.00 Lipoic acid 0.20 0.20 0.20 0.20 -- -- -- Phenol
red 10.00 10.00 10.00 10.00 10.00 10.00 10.00 Sodium pyruvate
110.00 110.00 110.00 110.00 -- -- -- Sodium succinate -- -- -- --
-- -- -- Succinic acid -- -- -- -- -- -- -- AMINO ACIDS: L-Alanine
25.00 25.00 25.00 25.00 -- -- -- L-Arginine 105.00 -- 105.00 -- --
-- -- L-Arginine-HCl -- 126.64 -- 126.64 126.00 126.00 126.00
L-Asparagine-H.sub.2O 50.00 50.00 50.00 50.00 -- -- -- L-Aspartic
acid 30.00 30.00 30.00 30.00 -- -- -- L-Cystine 24.00 -- 24.00 --
24.00 24.00 24.00 L-Cystine-2HCl -- 31.28 -- 31.28 -- -- --
L-Cysteine-HCl.H.sub.2O 100.00 100.00 100.00 100.00 -- -- --
L-Glutamic acid 75.00 75.00 75.00 75.00 -- -- -- L-Glutamine 292.00
292.00 292.00 292.00 292.00 -- -- Glycine 50.00 50.00 50.00 50.00
-- -- -- L-Histidine 31.00 -- 31.00 -- -- -- --
L-Histidine-HCl.H.sub.2O -- 42.00 -- 42.00 42.00 42.00 42.00
L-Isoleucine 52.40 52.40 52.40 52.40 52.00 52.00 52.00 L-Leucine
52.40 52.40 52.40 52.40 52.00 52.00 52.00 L-Lysine 58.00 -- 58.00
-- -- -- -- L-Lysine-HCl -- 72.50 -- 72.50 72.50 72.50 72.50
L-Methionine 15.00 15.00 15.00 15.00 15.00 15.00 15.00
L-Phenylalanine 32.00 32.00 32.00 32.00 32.00 32.00 32.00 L-Proline
40.00 40.00 40.00 40.00 -- -- -- L-Serine 25.00 25.00 25.00 25.00
-- -- -- L-Threonine 48.00 48.00 48.00 48.00 48.00 48.00 48.00
L-Tryptophan 10.00 10.00 10.00 10.00 10.00 10.00 10.00 L-Tyrosine
36.00 -- 36.00 -- 36.00 36.00 36.00 L-Tyrosine-2Na.2H.sub.2O --
51.90 -- 51.90 -- -- -- D-Valine -- -- -- -- 92.00 -- -- L-Valine
46.00 46.00 46.00 46.00 -- 46.00 46.00 VITAMINS: L-Ascorbic acid
50.00 50.00 50.00 50.00 -- -- -- Biotin 0.10 0.10 0.10 0.10 -- --
-- D-Ca pantothenate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Choline
bitartrate -- -- -- -- -- -- -- Choline chloride 1.00 1.00 1.00
1.00 1.00 1.00 1.00 Folic acid 1.00 1.00 1.00 1.00 1.00 1.00 1.00
i-Inositol 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Niacinamide 1.00 1.00
1.00 1.00 1.00 1.00 1.00 Pyridoxal-HCl 1.00 1.00 1.00 1.00 1.00
1.00 1.00 Riboflavin 0.10 0.10 0.10 0.10 0.10 0.10 0.10
Thiamine-HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Vitamin B.sub.12
1.36 1.36 1.36 1.36 -- -- -- RIBONUCLEOSIDES: Adenosine -- -- 10.00
10.00 -- -- -- Cytidine -- -- 10.00 10.00 -- -- -- Guanosine -- --
10.00 10.00 -- -- -- Uridine -- -- 10.00 10.00 -- -- --
DEOXYRIBONUCLEOSIDES: 2' Deoxyadenosine -- -- 10.00 10.00 -- -- --
2' Deoxycytidine-HCl -- -- 11.00 11.00 -- -- -- 2' Deoxyguanosine
-- -- 10.00 10.00 -- -- -- Thymidine -- -- 10.00 10.00 -- -- --
330-1430 410-1700 320-1890 320-1096 410-2400 320-1097 410-2500 10X
Liquid Powder 1X Liquid 1X Liquid Powder 1X Liquid Powder COMPONENT
mg/L mg/L mg/L mg/L mg/L mg/L mg/L INORGANIC SALTS: CaCl.sub.2
(anhyd.) 2000.00 200.00 200.00 200.00 200.00 200.00 200.00 KCl
400.00 400.00 400.00 400.00 400.00 400.00 400.00 MgSO.sub.4
(anhyd.) -- 97.67 -- -- 97.67 -- 97.67 MgSO.sub.4.7H.sub.2O 2000.00
-- 200.00 200.00 -- 200.00 -- NaCl 68000.00 6800.00 6800.00 6800.00
6800.00 6800.00 6800.00 NaHCO.sub.3 -- -- 2200.00 1500.00 --
2200.00 -- NaH.sub.2PO.sub.4.H.sub.2O.sup.a 1400.00 140.00 140.00
140.00 140.00 -- -- OTHER COMPONENTS: D-Glucose 10000.00 1000.00
1000.00 1000.00 1000.00 1000.00 1000.00 HEPES -- -- -- -- -- -- --
Lipoic acid -- -- -- -- -- -- -- Phenol red 100.00 6.00 10.00 10.00
10.00 10.00 10.00 Sodium pyruvate -- -- -- -- -- -- -- Sodium
succinate -- 100.00 -- -- -- -- -- Succinic acid -- 75.00 -- -- --
-- -- AMINO ACIDS: L-Alanine -- -- -- -- -- -- -- L-Arginine -- --
-- -- -- -- -- L-Arginine-HCl 1260.00 126.00 126.00 126.00 126.00
126.00 126.00 L-Asparagine-H.sub.2O -- -- -- -- -- -- -- L-Aspartic
acid -- -- -- -- -- -- -- L-Cystine 240.00 -- 24.00 -- -- 24.00 --
L-Cystine-2HCl -- 31.00 -- 31.00 31.29 -- 31.29
L-Cysteine-HCl.H.sub.2O -- -- -- -- -- -- -- L-Glutamic acid -- --
-- -- -- -- -- L-Glutamine -- -- -- -- 292.00 292.00 292.00 Glycine
-- -- -- -- -- -- -- L-Histidine -- -- -- -- -- -- --
L-Histidine-HCl.H.sub.2O 420.00 42.00 42.00 42.00 42.00 42.00 42.00
L-Isoleucine 520.00 52.00 52.00 52.00 52.00 52.00 52.00 L-Leucine
520.00 52.00 -- 52.00 -- 52.00 52.00 L-Lysine -- -- -- -- -- -- --
L-Lysine-HCl 725.00 72.50 72.50 72.50 -- 72.50 72.50 L-Methionine
150.00 15.00 15.00 15.00 -- 15.00 15.00 L-Phenylalanine 320.00
32.00 32.00 32.00 32.00 32.00 32.00 L-Proline -- -- -- -- -- -- --
L-Serine -- -- -- -- -- -- -- L-Threonine 480.00 48.00 48.00 48.00
48.00 48.00 48.00 L-Tryptophan 100.00 10.00 10.00 10.00 10.00 10.00
10.00 L-Tyrosine 360.00 36.00 36.00 -- -- 36.00 --
L-Tyrosine-2Na.2H.sub.2O -- -- -- 51.90 51.90 -- 51.90 D-Valine --
-- -- -- -- -- -- L-Valine 460.00 46.00 46.00 46.00 46.00 46.00
46.00 VITAMINS: L-Ascorbic acid -- -- -- -- -- -- -- Biotin -- --
-- -- -- -- -- D-Ca pantothenate 10.00 1.00 1.00 1.00 1.00 1.00
1.00 Choline bitartrate -- 1.80 -- -- -- -- -- Choline chloride
10.00 1.00 1.00 1.00 1.00 1.00 1.00 Folic acid 10.00 1.00 1.00 1.00
1.00 1.00 1.00 i-Inositol 20.00 2.00 2.00 2.00 2.00 2.00 2.00
Niacinamide 10.00 1.00 1.00 1.00 1.00 1.00 1.00 Pyridoxal-HCl 10.00
1.00 1.00 1.00 1.00 1.00 1.00 Riboflavin 1.00 0.10 0.10 0.10 0.10
0.10 0.10 Thiamine-HCl 10.00 1.00 1.00 1.00 1.00 1.00 1.00 Vitamin
B.sub.12 -- -- -- -- -- -- -- RIBONUCLEOSIDES: Adenosine -- -- --
-- -- -- -- Cytidine -- -- -- -- -- -- -- Guanosine -- -- -- -- --
-- -- Uridine -- -- -- -- -- -- -- DEOXYRIBONUCLEOSIDES: 2'
Deoxyadenosine -- -- -- -- -- -- -- 2' Deoxycytidine-HCl -- -- --
-- -- -- -- 2' Deoxyguanosine -- -- -- -- -- -- -- Thymidine -- --
-- -- -- -- -- .sup.1Eagle, H. (1959) Science, 130, 432.
.sup.2Nature, New Biology (1971) 230, 310. .sup.aOriginal formula
lists this component as NaH.sub.2PO.sub.4.2H.sub.2O.
[0071]
6 F-10 Nutrient Mixture (Ham).sup.1 320-1550 330-1955 380-2390
430-1200 1X Liquid 10X Liquid 1X Liquid Powder COMPONENT mg/L mg/L
mg/L mg/L INORGANIC SALTS: CaCl.sub.2 (anhyd.) -- -- -- 33.29
CaCl.sub.2.2H.sub.2O 44.10 441.00 44.10 --
CuSO.sub.4.5H.sub.2O.sup.a 0.0025 0.025 0.0025 0.0025
FeSO.sub.4.7H.sub.2O 0.834 8.34 0.834 0.834 KCl 285.00 2850.00
285.00 285.00 KH.sub.2PO.sub.4 83.00 830.00 83.00 83.00 MgCl.sub.2
(anhyd.) -- -- -- -- MgCl.sub.2.6H.sub.2O -- -- -- -- MgSO.sub.4
(anhyd.) -- -- -- 74.64 MgSO.sub.4.7H.sub.2O 152.80 1528.00 152.80
-- NaCl 7400.00 74000.00 5950.00 7400.00 NaHCO.sub.3 1200.00 --
1200.00 -- Na.sub.2HPO.sub.4 (anhyd.) -- -- -- 153.70
Na.sub.2HPO.sub.4.7H.sub.2O 290.00 2900.00 290.00 --
ZnSO.sub.4.7H.sub.2O 0.0288 0.288 0.0288 0.0288 OTHER COMPONENTS:
D-Glucose 1100.00 11000.00 1100.00 1100.00 HEPES -- -- 5958.00 --
Hypoxanthine 4.00 40.00 4.00 -- Hypoxanthine-Na -- -- -- 4.68
Linoleic acid -- -- -- -- Lipoic acid 0.20 2.00 0.20 0.20 Phenol
red 1.20 12.00 1.20 1.20 Putrescine-2HCl -- -- -- -- Sodium
pyruvate 110.00 1100.00 110.00 110.00 Thymidine 0.70 7.00 0.70 0.70
AMINO ACIDS: L-Alanine 9.00 90.00 9.00 9.00 L-Arginine-HCl 211.00
2110.00 211.00 211.00 L-Asparagine-H.sub.2O 15.01 150.10 15.01
15.01 L-Aspartic acid 13.00 130.00 13.00 13.00 L-Cysteine 25.00
250.00 25.00 25.00 L-Cysteine-HCl.H.sub.2O -- -- -- -- L-Glutamic
acid 14.70 147.00 14.70 14.70 L-Glutamine 146.00 1460.00 146.00
146.00 Glycine 7.51 75.10 7.51 7.51 L-Histidine-HCl.H.sub.2-
O.sup.b 23.00 230.00 23.00 23.00 L-Isoleucine 2.60 26.00 2.60 2.60
L-Leucine 13.00 130.00 13.00 13.00 L-Lysine-HCl 29.00 290.00 29.00
29.00 L-Methionine 4.48 44.80 4.48 4.48 L-Phenylalanine 5.00 50.00
5.00 5.00 L-Proline 11.50 115.00 11.50 11.50 L-Serine 10.50 105.00
10.50 10.50 L-Threonine 3.57 35.70 3.57 3.57 L-Tryptophan 0.60 6.00
0.60 0.60 L-Tyrosine 1.81 18.10 1.81 -- L-Tyrosine-2Na.2H.sub.2O --
-- -- 2.61 L-Valine 3.50 35.00 3.50 3.50 VITAMINS: Biotin 0.024
0.24 0.024 0.024 D-Ca pantothenate.sup.c 0.715.sup.d 7.15 0.715
0.715.sup.d Choline chloride 0.698 6.98 0.698 0.698 Folic acid 1.32
13.20 1.32 1.32 i-Inositol 0.541 5.41 0.541 0.541 Niacinamide 0.615
6.15 0.615 0.615 Pyridoxine HCl 0.206 2.06 0.206 0.206 Riboflavin
0.376 3.76 0.376 0.376 Thiamine HCl 1.00 10.00 1.00 1.00 Vitamin
B.sub.12 1.36 13.60 1.36 1.36 .sup.1Ham R. G. (1963) Exp. Cell.
Res. 29, 515. .sup.aTissue Culture Standards Committee lists this
as CuSO.sub.4.6H.sub.2O .sup.bOriginal formula lists
L-Histidine-HCl at 21.0 mg/L. .sup.cValues established by the
Tissue Culture Committee. .sup.dVaries from Tissue Culture
Standards Committee value of 0.238 mg/L.
[0072]
7 F-12 Nutrient Mixture (Ham).sup.1 320-1765 430-1700 1X Liquid
Powder COMPONENT mg/L mg/L INORGANIC SALTS: CaCl.sub.2 (anhyd.) --
33.22 CaCl.sub.2.2H.sub.2O 44.00 -- CuSO.sub.4.5H.sub.2O 0.0025
0.0025 FeSO.sub.4.7H.sub.2O 0.834 0.834 KCl 223.60 223.60
MgCl.sub.2 (anhyd.) -- 57.22 MgCl.sub.2.6H.sub.2O 122.00 -- NaCl
7599.00 7599.00 NaHCO.sub.3 1176.00 -- Na.sub.2HPO.sub.4 (anhyd.)
-- 142.04 Na.sub.2HPO.sub.4.7H.sub.2O 268.00 --
ZnSO.sub.4.7H.sub.2O 0.863 0.863 OTHER COMPONENTS: D-Glucose
1802.00 1802.00 Hypoxanthine 4.10 -- Hypoxanthine (sodium salt) --
4.77 Linoleic acid 0.084 0.084 Lipoic acid 0.21 0.21 Phenol red
1.20 1.20 Putreacine-2HCl 0.161 0.161 Sodium pyruvate 110.00 110.00
Thymidine 0.73 0.73 AMINO ACIDS: L-Alanine 8.90 8.90 L-Arginine-HCl
211.00 211.00 L-Asparagine-H.sub.2O 15.01 15.01 L-Aspartic acid
13.30 13.30 L-Cysteine-HCl.H.sub.2O 35.12 35.12 L-Glutamic acid
14.70 14.70 L-Glutamine 146.00 146.00 Glycine 7.50 7.50
L-Histidine-HCl.H.sub.2O 20.96 20.96 L-Isoleucine 3.94 3.94
L-Leucine 13.10 13.10 L-Lysine-HCl 36.50 36.50 L-Methionine 4.48
4.48 L-Phenylalanine 4.96 4.96 L-Proline 34.50 34.50 L-Serine 10.50
10.50 L-Threonine 11.90 11.90 L-Tryptophan 2.04 2.04 L-Tyrosine
5.40 -- L-Tyrosine-2Na.2H.sub.2O -- 7.78 L-Valine 11.70 11.70
VITAMINS: Biotin 0.0073 0.0073 D-Ca pantothenate 0.48 0.48 Choline
chloride 13.96 13.96 Folic acid 1.30 1.30 i-Inositol 18.00 18.00
Niacinamide 0.037 0.037 Pyridoxine-HCl 0.062 0.062 Riboflavin 0.038
0.038 Thiamine-HCl 0.34 0.34 Vitamin B.sub.12 1.36 1.36 .sup.1Ham,
R. G. (1965) Proc. Nat. Acad. Sci. 53, 288.
[0073]
8 RPMI Medium 1630.sup.1 320-1855 1X Liquid COMPONENT mg/L
INORGANIC SALTS: Ca(NO.sub.3).sub.2.4H.sub.2O 100.00 KCl 400.00
MgSO.sub.4.7H.sub.2O 100.00 NaCl 6000.00
Na.sub.2HPO.sub.4.7H.sub.2O 2835.00 OTHER COMPONENTS: D-Glucose
2500.00 Glutathione (reduced) 10.00 Phenol red 5.00 AMINO ACIDS:
L-Arginine 200.00 L-Asparagine 30.00 L-Aspartic acid 30.00
L-Cystine 100.00 L-Glutamic acid 80.00 L-Glutamine 300.00 Glycine
15.00 L-Histidine 35.00 L-Isoleucine 50.00 L-Leucine 50.00 L-Lysine
HCl 60.00 L-Methionine 15.00 L-Phenylalanine 30.00 L-Proline 30.00
L-Serine 50.00 L-Threonine 50.00 L-Tryptophan 10.00 L-Tyrosine
30.00 L-Valine 40.00 VITAMINS: Biotin 0.20 D-Ca pantothenate 3.00
Choline chloride 3.00 Folic acid 2.00 i-Inositol 5.00 Niacinamide
2.50 Para-aminobenzoic acid 0.50 Pyridoxine-HCl 2.00 Riboflavin
0.50 Thiamine-HCl 5.00 Vitamin B.sub.12 0.05 .sup.1Moore, G. E. and
Kitamura, H. (1968) N. Y. State Journal of Medicine 68, 2054.
[0074]
9 RPMI Media 1640.sup.1 320-1870 320-1875 330-2511 380-2400
430-1800 430-3200 430-3400 320-1835 320-1877 1X Liquid 1X Liquid
10X Liquid 1X Liquid Powder Powder Powder 1X Liquid 1X Liquid
COMPONENT mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L INORGANIC
SALTS: Ca(NO.sub.3).sub.2.4H.sub.2O 100.00 100.00 1000.00 100.00
100.00 100.00 100.00 100.00 100.00 KCl 400.00 400.00 4000.00 400.00
400.00 400.00 400.00 400.00 400.00 MgSO.sub.4 (anhyd.) -- -- -- --
48.84 48.84 48.84 -- -- MgSO.sub.4.7H.sub.2O 100.00 100.00 1000.00
100.00 -- -- -- 100.00 100.00 NaCl 6000.00 6000.00 60000.00 5300.00
6000.00 6000.00 5850.00 6000.00 6000.00 NaHCO.sub.3 2000.00 2000.00
-- 2000.00 -- -- -- 2000.00 2000.00 Na.sub.2HPO.sub.4 (anhyd.) --
-- -- -- 800.00 800.00 800.00 -- -- Na.sub.2HPO.sub.4.7H.sub.2O
1512.00 1512.00 15120.00 1512.00 -- -- -- 1512.00 -- OTHER
COMPONENTS: D-Glucose 2000.00 2000.00 20000.00 2000.00 2000.00
2000.00 2000.00 2000.00 2000.00 Glutathione (reduced) 1.00 1.00
10.00 1.00 1.00 1.00 1.00 1.00 1.00 HEPES -- -- -- 5958.00 -- --
5957.50 -- -- Phenol red 5.00 5.00 50.00 5.00 5.00 -- 5.00 -- 5.00
AMINO ACIDS: L-Arginine 200.00 200.00 2000.00 200.00 200.00 200.00
200.00 200.00 200.00 L-Asparagine 50.00 50.00 500.00 50.00 50.00
50.00 50.00 50.00 50.00 L-Aspartic acid 20.00 20.00 200.00 20.00
20.00 20.00 20.00 20.00 20.00 L-Cystine 50.00 50.00 500.00 50.00 --
-- -- 50.00 50.00 L-Cystine-2HCl -- -- -- -- 65.15 65.15 65.15 --
-- L-Glutamic acid 20.00 20.00 200.00 20.00 20.00 20.00 20.00 20.00
20.00 L-Glutamine -- 300.00 3000.00 300.00 300.00 300.00 300.00
300.00 300.00 Glycine 10.00 10.00 100.00 10.00 10.00 10.00 10.00
10.00 10.00 L-Histidine 15.00 15.00 150.00 15.00 15.00 15.00 15.00
15.00 15.00 L-Hydroxyproline 20.00 20.00 200.00 20.00 20.00 20.00
20.00 20.00 20.00 L-Isoleucine 50.00 50.00 500.00 50.00 50.00 50.00
50.00 50.00 50.00 L-Leucine 50.00 50.00 500.00 50.00 50.00 50.00
50.00 50.00 50.00 L-Lysine-HCl 40.00 40.00 400.00 40.00 40.00 40.00
40.00 40.00 40.00 L-Methionine 15.00 15.00 150.00 15.00 15.00 15.00
15.00 15.00 15.00 L-Phenylalanine 15.00 15.00 150.00 15.00 15.00
15.00 15.00 15.00 15.00 L-Proline 20.00 20.00 200.00 20.00 20.00
20.00 20.00 20.00 20.00 L-Serine 30.00 30.00 300.00 30.00 30.00
30.00 30.00 30.00 30.00 L-Threonine 20.00 20.00 200.00 20.00 20.00
20.00 20.00 20.00 20.00 L-Tryptophan 5.00 5.00 50.00 5.00 5.00 5.00
5.00 5.00 5.00 L-Tyrosine 20.00 20.00 200.00 20.00 -- -- -- 20.00
20.00 L-Tyrosine-2Na.2H.sub.2O -- -- -- -- 28.83 28.83 28.83 -- --
L-Valine 20.00 20.00 200.00 20.00 20.00 20.00 20.00 20.00 20.00
VITAMINS: Biotin 0.20 0.20 2.00 0.20 0.20 0.20 0.20 0.20 0.20 D-Ca
pantothenate 0.25 0.25 2.50 0.25 0.25 0.25 0.25 0.25 0.25 Choline
chloride 3.00 3.00 30.00 3.00 3.00 3.00 3.00 3.00 3.00 Folic acid
1.00 1.00 10.00 1.00 1.00 1.00 1.00 1.00 1.00 i-Inositol 35.00
35.00 350.00 35.00 35.00 35.00 35.00 35.00 35.00 Niacinamide 1.00
1.00 10.00 1.00 1.00 1.00 1.00 1.00 1.00 Para-aminobenzoic acid
1.00 1.00 10.00 1.00 1.00 1.00 1.00 1.00 1.00 Pyridoxine-HCl 1.00
1.00 10.00 1.00 1.00 1.00 1.00 1.00 1.00 Riboflavin 0.20 0.20 2.00
0.20 0.20 0.20 0.20 0.20 0.20 Thiamine-HCl 1.00 1.00 10.00 1.00
1.00 1.00 1.00 1.00 1.00 Vitamin B.sub.12 0.005 0.005 0.05 0.005
0.005 0.005 0.005 0.005 0.005 .sup.1Moore, G. E., Gerner, R. E.,
and Franklin, H. A. (1967) J.A.M.A. 199, 519.
[0075] The serum component may be present in the culture in an
amount of at least 1% (v/v) to 50% (v/v). The preferred range will
depend on whether or not serum is being used alone or is, at least
in part, replaced by a serum replacement. When using no serum
replacement, the serum concentration may be preferably in the
neighborhood of 10 to 30% (v/v). The third component,
corticosteroid, may be present in an amount of from 10.sup.-7 M to
10.sup.-4 M, and is preferably present in an amount of from
5.times.10.sup.-6 to 5.times.10.sup.-5 M. Alternatively, the serum
component can be replaced by any of several standard serum
replacement mixtures which typically include insulin, albumin, and
transferrin, lecithin, selenium or cholesterol. See, Migliaccio et
al, Exp. Hematol. (1990) 18:1049-1055, Iscove et al, Exp. Cell Res.
(1980) 126:121-126, and Dainiak et al, J. Clin. Invest. (1985)
76:1237-1242.
[0076] In addition to supporting the proliferation of human
hematopoietic stem cells, the applications of these same conditions
leads to the controlled production or depletion of specific lineage
of blood cells. The inventors have discovered that when IL-3 and
Epo, with or without GM-CSF, are used as described above one
obtains lineage specific development of red blood cells. The
inventors have also observed that T and B lymphocytes are lost from
these cultures, during the same period of time in which the myeloid
progenitor and cell mass is increasing. The inventors have also
observed that l ukemic cells are lost from these cultures over
time.
[0077] The inventors also observed that with the cultures of the
invention T and B lymphocytes are lost over time. As noted above,
there are several T-cell-derived diseases and therapeutic concerns.
For example, the autoimmune deficiency diseases (e.g., AIDS) result
because of abnormal T-cell function caused by direct viral
infection. Since this order results as a direct infection of the
mature T-cell, and is not derived from defective hematopoietic stem
or progenitor cells, selective eradication of the mature T-cells
has notable potential therapeutic benefit.
[0078] T-cell depletion has other applications as well. A limiting
factor to the improved success of allogeneic bone marrow transplant
is T-cell mediated. Depletion of T-cells from a stem/progenitor
cell population prior to allogeneic transplant would enhance the
reingraftment success by reducing the T-cell mediated graft versus
host-rejection response.
[0079] The inventors have discovered that the present methods,
including the present culture media conditions, which allow for the
in vitro replication and differentiation of human stem and
hematopoietic progenitor cells do not allow for maintenance of all
hematopoietic cell classes. Although in the present methods and
composition, human stem and hematopoietic progenitor cells are
capable of in vitro replication and differentiation, human T-cells
and B-cells, a major class of peripheral blood cells, do not
proliferate or maintain viability in these in vitro culture
conditions.
[0080] More particularly, T-cells require, among other factors, the
growth factor interleukin-2 (IL-2) to remain viable. T-cells grown
without such proper support die in approximately 3 to 4 days in a
medium substantially free of IL-2. As a result, over a period of
time when the viability and proliferative capacity of human stem
and hematopoietic progenitor cells are maintained in accordance
with the invention, the human T-cells contained in the human
hematopoietic mononuclear cell population die, if the medium is
substantially free of IL-2.
[0081] In accordance with this embodiment of the invention, a mixed
human hematopoietic mononuclear cell fraction can be effectively
depleted of T-cells and B-cells, under conditions which allow for
expansion of stem and progenitor cells.
[0082] This method of selective T-cell depletion of human
hematopoietic cell populations has notable therapeutic value as
noted above. These include the following two applications:
[0083] 1. Supportive treatment alone, or when used as adjuvant
therapy, for curative treatment of AIDS and related T-cell diseases
resulting from the dysfunction of the mature T-cell because of
viral infection or other T-cell-specific functional disruption. The
present culture conditions can be used to deplete disease-causing
T-cells, while allowing for the survival, with or without their
expansion, of human hematopoietic stem and/or progenitor cells. The
T-cell-depleted hematopoietic cell population can then be used for
reingraftment of patient bone marrow. The reestablished marrow will
then produce anew, normal T-cell population. In its application to
AIDS therapy, this procedure itself would not serve to necessarily
eradicate the HIV from the patient, and reinfection of the newly
developed T-cell in vivo is likely. Accordingly, this therapy would
be considered as supportive, but, if used with other
virus-eradication procedures, this procedure is operative for
curative treatment protocols as well.
[0084] 2. Allogeneic bone marrow transplant whereby a human
hematopoietic stem/progenitor cell population is depleted of viable
T-cells and then used to reestablish the hematopoietic system in a
recipient individual. The depletion of the T-cell population will
increase the prospect of successful reingraftment by decreasing the
graft versus host rejection process.
[0085] The present method of T-cell and B-cell depletion should be
contrasted with methods for isolating and purifying progenitor
cells (see, e.g., U.S. Pat. No. 5,061,620 as representative). The
reported methods do not provide a method for the long term culture
of viable and replicating stem cells, while the present method
affords just such a result.
[0086] It should be understood that the present method for
controlling the lineage development in a human hematopoietic tissue
system may be practiced in conjunction with genetic transformation
of at least a portion of the stem cells in the hematopoietic tissue
system.
[0087] In another embodiment, the present invention provides a
method for assaying the effect of a substance or substances and/or
physical condition on a hematopoietic cell culture or the process
of hematopoiesis. In accordance with this embodiment, one may add
to a cell culture, carried out in accordance with the invention, at
least one substance suspected of having an affect, which may be
either beneficial or detrimental, on the cell culture. One may then
compare the cell culture state obtained in the absence of the
substance being tested to the cell culture state obtained in the
presence of the substance.
[0088] Compounds or substances which may be tested include those
which are expected to exert some effect on the hematopoietic
system. Such compounds include, for example, hematopoietic growth
factors, drugs, hormones, etc.
[0089] It should be understood that the present assay also permits
the determination of the effect of substances endogenously produced
by the present hematopoietic system. The effect of an endogenously
produced substance may be assayed by adding to the medium a
compound which either reduces the effective concentration of the
endogenously produced substance and/or inhibits the action of the
endogenously produced substance. Examples of such compounds include
monoclonal antibodies, which bind to and neutralize endogenously
produced growth factors, and antagonists, which bind to and block
growth factor receptors on the surface of cells.
[0090] The present assay may also be used to ascertain the effect
of physical conditions on the hematopoietic system or hematopoietic
process. Such conditions include, for example, temperature,
pressure, light intensity, gravity, etc. The effect of temperature,
pressure, and light intensity may be determined by varying these
parameters using conventional techniques and apparatus, such as
heaters, refrigerators, pressurized or reduced-pressure chambers,
and light sources. The effect of gravity may be determined by,
e.g., carrying out the present culture in a zero-gravity
environment, such as the space shuttle. The present assay may also
be used to determine the effect of the particular configuration of
the cell culture chamber, such as the nature of the surface which
supports the adherent cell population.
[0091] Of course, it should be understood that the present assay is
not limited to determining the effect of a single chemical
substance or physical condition but may be used to detect the
effect of the combined action of any number of chemical substances
and/or physical conditions.
[0092] The parameters which can be monitored in carrying out the
present assay include the cell population profile of the
hematopoietic cell culture, the total cell population, the relative
population of any particular type of cell, the presence, absence,
or concentration of any other substance in the medium being removed
from the culture, the consumption of nutrients, the morphology of
any or all of the particular cell types present in the culture, the
lifetime and duration of the culture, and the kinetics of
hematopoiesis.
[0093] Thus, the present invention provides a functioning in vitro
human hematopoietic tissue system which may serve as a model for
the study of the naturally occurring in vivo hematopoietic system
and the process of hematopoiesis. Accordingly, the effect of any
chemical substance and/or physical condition on the hematopoietic
system or the process of hematopoiesis may be determined by the
present assay. It should be recognized that the present assay
exhibits a significant advantage in that the effects of (i) slow
acting substances and/or conditions or (ii) substances and/or
conditions for which the effect exhibits a lag phase may be readily
determined, because the present system provides a long term
functioning hematopoietic tissue system.
[0094] It should also be understood that the present assay may be
carried out when at least a portion of the stem cells in the human
hematopoietic tissue system have been genetically transformed. Such
genetic transformation may be used to introduce genetic markers
useful for the subsequent identification of cells derived from the
transformed stem cells. In addition, such genetic transformation
may also serve as a method for introducing into the medium the
chemical substance to be studied. Thus, when stem cells are
transformed with a gene encoding for a particular substance and the
appropriate regulatory sequences, the production of the substance
by either the stem cell or a cell derived therefrom will provide a
constant source of the substance.
[0095] In another embodiment, the present invention provides an
improved method of bone marrow transplantation. Thus, by culturing
bone marrow tissue according to the present method may of the
drawbacks attendant to conventional bone marrow transplants may be
avoided.
[0096] Thus, as noted above, the present method of bone marrow
transplantation may be advantageously applied in situations in
which the bone marrow tissue has been previously removed from a
patient, before the patient is subjected to either chemotherapy or
radiation therapy for the treatment of cancer, and is then
implanted in the same patient after completion of the session of
therapy. Since the present methods permit the expansion of the
hematopoietic culture, a smaller quantity of tissue may be removed
form the patient prior to therapy. In addition, since culture of
the bone marrow tissue according to the present method results in
the depletion and extinction of malignant cells, implantation of
the tissue cultured according to the present method poses a reduced
risk of reintroducing malignant cells which may have metastasized
into the bone marrow.
[0097] In the setting of allogeneic bone marrow transplants, the
present method also exhibits distinct advantages. Thus, because
culturing according to the present method, using medium
substantially free of IL-2, results in a bone marrow culture
substantially free of T-cells and B-cells and since such cells are
principally involved in graft versus host disease, implantation of
allogeneic bone marrow tissue cultured according to the present
method poses a reduced risk of graft versus host disease.
[0098] It should be stressed that these advantages are uniquely
afforded by the present invention. Thus, it is the ability of the
present culture techniques to maintain and/or expand a viable and
functioning in vitro hematopoietic tissue system for a time
sufficient to effect the depletion of malignant cells and/or
T-cells and B-cells that enables the implantation of bone marrow
tissue substantially free of malignant cells and/or T-cells and
B-cells. Culturing bone marrow tissue, for a time sufficient to
effect such depletions, by conventional techniques would not result
in a functioning in vitro hematopoietic tissue system containing
viable stem cells.
[0099] The present method of bone marrow transplantation may be
carried out as follows: removing a tissue sample from a donor;
culturing said tissue sample according to the present method; and
implanting said cultured tissue in a donee. As noted above the
donor and donee may be the same or different.
[0100] The tissue sample may be obtained from the donor according
to conventional methods using conventional apparatus. Apparatus for
and methods of bone marrow transplantation are disclosed in U.S.
Pat. Nos. 4,481,946 and 4,486,188, which are incorporated herein by
reference.
[0101] After the tissue sample has been obtained, it is then
cultured according to the present method, for a time sufficient to
achieve the desired expansion and/or cell depletion. The cultured
tissue may then be implanted in the donee, again, according to
conventional techniques.
[0102] It should be understood that the present method of bone
marrow transplantation may be used advantageously in conjunction
with genetic transformation of at least a portion of the stem cells
in the tissue to be implanted in the donee. The advantages of stem
cell transformation in gene therapy are discussed above. Thus, the
present method of bone marrow transplantation, when used in
conjunction with genetic transformation of stem cells in the
implanted tissue, represents an improved method of gene
therapy.
[0103] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only and are not intended to be limiting unless otherwise
specified.
EXAMPLES
Example 1
Medium Replacement
[0104] Materials and Methods:
[0105] Cells: Human bone marrow cells were obtained from
heparinized aspirates from the iliac crest of informed and
consenting individuals. The bone marrow was separated by a
Ficoll-Paque (Pharmacia, No. 17-0840-02) density gradient
centrifugation and the low density cells (<1.077 gm/cm.sup.3)
were collected and washed 3 times with Iscove's Modified Dulbecco's
Medium (IMDM). The cells were counted between the second and third
washes. The cells were then seeded onto 24-well tissue culture
plates (Costar No. 3524) in duplicate or triplicate at 1, 2, and
5.10.sup.6 cells/ml at 322 .mu.l/well.
[0106] Long-term culture conditions: The low density cells were
incubated in IMDM supplemented with 10% fetal calf serum (Hyclone
Laboratories), 10% horse serum (Hyclone Laboratories), 1%
penicillin/streptomycin (Sigma, 10,000 U/ml penicillin G and 10
mg/ml streptomycin, Cat. No. P3539), and 10.sup.-5 M hydrocortisone
(17-Hydroxy-corticosterone, Sigma, Cat. No. H0888) in a humidified
5% CO.sub.2/95% air atmosphere. The cultures were treated with one
of three medium exchange schedules, 100% daily medium exchange
(7/wk), 50% daily medium exchange (3.5/wk), or 50% biweekly medium
exchange (1/wk). Twice per week during the medium exchange, 50% of
the non-adherent cells were removed from each culture well and
counted using a hemocytometer. When the cells were removed for
counting (twice/week), all of the medium removed during feeding of
the 3.5/wk and 1/wk cultures was saved for cell counts and fresh
medium returned to the wells. The 7/wk cultures required saving 1/2
of the removed medium for cell counts, while centrifuging and
returning the non-adherent cells in the remaining 1/2 of the medium
removed. Fresh medium was then added to each well to replace the
medium removed for cell counts. On days when the cells were not
removed for counting, 100% or 50% of the medium was removed from
each of the 7/wk and 3.5/wk culture wells respectively, the cells
were centrifuged and returned to the original wells with additional
fresh medium.
[0107] Methylcellulose and morphologic assays: Once every other
week, the non-adherent cells removed for cell counts were plated in
methylcellulose in the presence of erythropoietin, GM-CSF, and
IL-3, and the Granulocyte Macrophage-Colony Forming Units (CFU-GM)
were enumerated. Aliquots of removed cells were cytocentrifuged,
stained with Wright-Giemsa, and differential cell counts were
performed.
[0108] Statistical analvsis: The biweekly cell production results
are expressed as the mean.+-.SEM from replicate cultures. The
probability of significant differences between groups of cultures
was determined by comparing the normalized cumulative cell
production values from the rapidly exchanged cultures (7/wk and
3.5/wk) to the matched control cultures (1/wk) using a paired
t-test. Statistical significance was taken at the 5% level.
[0109] Results:
[0110] Kinetics of nonadherent cell production: Nonadherent cell
production was examined both as a function of inoculum cell density
(over the range 1-5.multidot.10.sup.6 cells/ml) and medium exchange
rate. The medium exchange rate was varied from one medium volume
exchange per week, the traditional Dexter culture rate, to seven
medium volume exchanges per week. The biweekly number of cells
collected was normalized by dividing by the number of cells
inoculated per culture. At each medium exchange rate, the
normalized cell collection curves did not change significantly with
inoculum density. The cell production for the cultures maintained
at the three medium perfusion rates of 7/wk, 3.5/wk and 1/wk were
similar when normalized to the number of cells inoculated per
culture. Comparison of the final cumulative cell productions
between inoculum densities showed no significant differences, at
any of the three medium exchange rates (p>0.20 by a paired
t-test for all pairs of samples).
[0111] The medium exchange rate, in contrast, strongly influenced
the rate and longevity of cell production in these cultures. Cell
production of the cultures exchanged at 1/wk (control), 3.5/wk, and
7/wk all decayed over the first few weeks. Differences in culture
productivity, however, became apparent after week 3 in culture.
Between weeks 3 to 10, the cell production was constant in the 7/wk
cultures, constant at a lower level in the 1/wk cultures, but
increased exponentially in the 3.5/wk cultures. After weeks 10 to
12, cell production declined in all cultures until culture
termination. Results for the 1/wk exchanged cultures are equivalent
to those commonly observed in traditional human Dexter cultures in
a variety of systems, whereas the rapidly exchanged cultures of 3.5
and 7/wk showed increased cell productivity when compared to
previous optimum culture methods. Cultures in which 1/2 of the
medium was exchanged daily (3.5/wk) maintained increased cell
production for substantially longer than either the control (1/wk)
or complete daily exchange (7/wk) cultures. Between weeks 3 and 9,
the number of nonadherent cells collected from the 3.5/wk exchanged
cultures increased exponentially with a doubling every 2.1
weeks.
[0112] The cell production under the 3.5/wk and 1/wk protocols can
be directly compared by plotting the cell production under the
3.5/wk exchange rate as a percentage of the production of the
cultures with an exchange rate of 1/wk. This comparison shows that
during the initial decay phase the cell production under the two
protocols is similar. However, between weeks 3.5 and 18, the cell
production under the 3.5/wk exchange rate is consistently
higher.
[0113] The proliferative potential of the cultures can be measured
by their ability to produce cells following the initial decay. The
normalized cumulative cell production following week 3 was
independent of the cell inoculation density for the medium exchange
rates of 7/wk, 3.5/wk. Cell production data from the cultures at
similar medium exchange rates were qualitatively and statistically
similar, and were therefore density averaged and combined to obtain
a larger statistical sample. The density averaged cumulative cell
production between weeks 3.5 and 20 was: 0.22 for the 7/wk; 0.40
for the 3.5/wk; and 0.15 for the 1/wk cultures. The increase in the
medium exchange rate from 1/wk to 7/wk thus increased the cell
production about 60% over the typical Dexter culture medium
exchange schedule. The 3.5/wk exchange rate resulted in almost
3-fold cumulative cell production increase compared to the 1/wk
Dexter protocol. Statistical analysis of these data using a paired
t-test, demonstrated significant differences between both the 7/wk
vs. 1/wk and the 3.5/wk vs. 1/wk at the 5% level of significance.
The medium exchange rate of 3.5/wk thus improves the cell
production rate over the traditional Dexter protocol of 1/wk.
[0114] Granulocyte-macrophage progenitor cell production:
Granulocyte-macrophage progenitor cell assays were performed from
replicates of a given medium perfusion schedule and inoculum
density (Table 1). The medium perfusion rate had a pronounced
effect on the number of granulocyte-macrophage progenitor cells
produced. The 3.5/wk medium exchange cultures showed the greatest
longevity in terms of progenitor cell production. These cultures
produced progenitors at a stable rate between weeks 4 and 18. The
optimum conditions in terms of progenitor cell production are the
cultures exchanged 3.5 times per week and inoculated at
5.multidot.10.sup.6 cells/ml. These cultures produced a significant
number of progenitor cells until week 20. Statistical analysis,
using a paired t-test, showed that the optimum medium exchange rate
cultures of 3.5/wk produced significantly more
granulocyte-macrophage progenitor cells after week 8 than did the
corresponding 7/wk and 1/wk cultures at all three inoculation
densities at the 1% level of significance. The number of progenitor
cells produced is important as it is an indirect measure of stem
cell renewal. Progenitor cells can only be present after several
weeks in culture by differentiation from an earlier cell,
presumably a stem cell, which is still present in culture. Thus,
these data suggest that more physiologic, rapid medium/serum
exchange rate and higher cell densities may have provided
conditions that supported some degree of stem cell renewal for five
months.
[0115] Nonadherent cell morphology: To determine whether the
prolonged hematopoiesis supported by the 3.5/wk cultures was
qualitatively different from the other cultures, the non-adherent
cells collected between weeks 10 and 19 were stained and typed
morphologically. At the exchange rates of 1/wk and 7/wk, the cells
produced were mostly macrophages by week 15 and thereafter (Table
2), which is similar to results from studies in other laboratories.
In contrast, the cultures perfused at a rate of 3.5 medium volumes
per week and seeded at 5.multidot.10.sup.6 cells/ml produced
granulocytes as well as macrophages through week 19.
[0116] Thus, rapid medium exchange effectively resulted in the
selective production of granulocytes throughout the cultures, a
feature which was not present without the application of rapid
medium exchange or perfusion. This result supports the hypothesis
that standard long term human hematopoietic culture conditions are
suboptimal, and that proper in vitro culture of hematopoietic cells
under the presently described conditions allows the production of
blood cells of diverse lineages.
10TABLE 1 The average number of nonadherent progenitor cells
removed from long term bone marrow cultures (LTBMCs) as a function
of the medium perfusion rate and inoculum density. 7/wk 3.5/wk 1/wk
5 .times. 10.sup.6 2 .times. 10.sup.6 1 .times. 10.sup.6 5 .times.
10.sup.6 2 .times. 10.sup.6 1 .times. 10.sup.6 5 .times. 10.sup.6 2
.times. 10.sup.6 1 .times. 10.sup.6 Week per ml per ml per ml per
ml per ml per ml per ml per ml per ml 2 237 .+-. 27 11 .+-. 3.3 106
.+-. 5 120 .+-. 16 132 .+-. 7.9 167 .+-. 13 368 .+-. 29 94 .+-.
20.8 335 .+-. 46 4 149 .+-. 21 101 .+-. 5.1 104 .+-. 10 93 .+-. 10
37 .+-. 5.6 20 .+-. 0 21 .+-. 1.3 2 .+-. 0 8 .+-. 4.4 6 47.7 .+-. 7
12 .+-. 2.5 8 .+-. 0 17 .+-. 3 6 .+-. 4.1 5 .+-. 2.7 13 .+-. 5.1 1
.+-. 0 1 .+-. 0 8 40 .+-. 3 0 4 .+-. 0 38 .+-. 6 24 .+-. 2.7 10
.+-. 3 34 .+-. 7.4 0 0 10 0 0 0 28 .+-. 8.3 10 .+-. 2.9 5 .+-. 1.3
8 .+-. 2.3 2 .+-. 2.3 0 12.5 0 6 .+-. 2.3 0 8 .+-. 2.3 0 0 0 0 0 14
0 0 0 22 .+-. 6.4 6 .+-. 1.3 2.5 .+-. 1.2 3 .+-. 1.3 0 0 16 6 .+-.
2.2 0 0 24 .+-. 7.6 4 .+-. 1.7 2 .+-. 1.3 9 .+-. 3.6 0 0 18 0 0 0
24 .+-. 6.3 4 .+-. 1.3 0 0 0 0 20 0 0 0 5 .+-. 0 4 .+-. 0 3 .+-. 0
1 .+-. 0 0 0 22 2 .+-. 1.3 0 0 4 .+-. 1.3 10 .+-. 3 0 0 0 0 10-22*
8 .+-. 3.5 6 .+-. 2.3 0 115 .+-. 32.2 40 .+-. 11.2 12.5 .+-. 3.8 21
.+-. 7.2 2 .+-. 7 0 Replicate samples at each medium perfusion rate
and inoculum density were pooled and are each tabulated as one mean
.+-. SEM. *Cumulative CFU-GM production after week 8 is
statistically greater in the 3.5/wk cultures than the corresponding
cultures perfused at 7/wk or 1/wk at all inoculum densities at the
1% level of significance.
[0117] Physical appearance: The medium exchange rate significantly
affected the physical appearance of the cultures. By 10 weeks in
culture, the 7/wk cultures had large number of adipose cells in the
stroma while the 3.5/wk cultures had few fat cells and the 1/wk
cultures never developed fat cells. At culture termination at 26
weeks, the stroma of the 7/wk cultures were composed of
approximately 20-30% fat cells while the 3.5/wk cultures still only
had a few fat cells. Adherent colony distribution also varied
between cultures with different medium perfusion rate. Adherent
colonies in the 3.5/wk cultures persisted longer than those in the
7/wk and 1/wk cultures.
11TABLE 2 Nonadherent cell morphology as a function of the medium
perfusion rate and inoculum density. 5 .times. 10.sup.6 per ml 2
.times. 10.sup.6 per ml 1 .times. 10.sup.6 per ml Medium % % %
perfusion myeloid myeloid myeloid rate weeks % M.PHI. % G
precursors % M.PHI. % G precursors % M.PHI. % G precursors 7/wk
10.4 25 57 18 57 32 11 52 34 14 13.4 49 34 17 92 5 3 63 22 15 15.4
66 19 16 79 19 2 54 17 29 19 93 5 1 96 3 1 100 0 0 3.5/wk 10.4 50
27 23 45 38 17 39 45 17 13.4 23 59 19 27 56 17 36 47 17 15.4 41 38
21 44 27 29 67 13 21 19 58 37 5 88 9 3 99 1 0 1/wk 10.4 59 21 20 60
11 29 ND ND ND 13.4 56 25 20 19 36 46 43 7 50 15.4 76 4 20 ND ND ND
46 39 15 19 100 0 0 100 0 0 100 0 0 Data are for pooled replicate
samples at each medium perfusion rate and inoculum density and are
shown as the percentage of macrophages (% M.PHI.), granulocytes
(mature granulocytes and bands, % G), and immature granulocytes
precursors (metamyelocytes, myelocytes, promyelocytes and
blasts).
Example 2
Medium Replacement Combined with Supplementation of Medium with
Hematopoietic Growth Factors
[0118] Materials and Methods:
[0119] Cells: Human bone marrow cells were obtained following
informed consent from heparinized aspirates of the iliac crest bone
marrow, under a protocol approved by the University of Michigan
Human Investigation Committee. The bone marrow was separated by a
Ficoll-Paque (Pharmacia) density gradient centrifugation and the
low density cells (<1.077 gm/cm.sup.3) were collected and washed
3 times with IMDM. The cells were counted between the second and
third washes. The cells were then seeded onto 0.6-well tissue
culture plates (Costar No. 3406) or collagen coated 6-well plates
(rat tail type 1 collagen, Biocoat. Collaborative Research Inc.
Cat. No. 40400) in duplicate 5.multidot.10.sup.6 cells/ml at 1.5
ml/well.
[0120] Culture medium: The medium used was IMDM (Gibco
Laboratories. Cat. No. 430-2200) containing 10% fetal calf serum
(Hyclone Laboratories), 10% horse serum (Hyclone Laboratories), 1%
penicillin/streptomycin (Sigma, 10,000 U/ml penicillin G and 10
mg/ml streptomycin, Cat. No. P3539), and 10.sup.-5M hydrocortisone
(17-Hydroxycorticosterone, Sigma, Cat. No. H0888).
[0121] Hematopoietic growth factors (HGH): Due to the frequent
culture supplementation via rapid medium exchange, hematopoietic
growth factors were added daily to the medium at approximately
{fraction (1/20)} of the concentrations found to promote maximal
colony formation in clonal assays. The concentrations used were 1
ng/ml of IL-3, 1 ng/ml of GM-CSF (Amgen Biologicals, Cat. No.
13050), 0.1 U/ml of Epo (Terry Fox Labs. Vancouver, Canada). In
this and all of the following examples, a unit of activity for any
given component is as defined by the supplier listed.
[0122] Hematopoietic progenitor cell assay: Nonadherent
hematopoietic cells removed from culture were counted and plated at
1.multidot.10.sup.5 cells/ml or fewer cells in methylcellulose.
GM-CSF and Epo were added to the methylcellulose at 20 ng/ml and 2
U/ml, respectively. The cells were plated in 24 well plates at 0.25
ml/well and incubated at 37.degree. C. for 14 days. The colonies
were then counted under an inverted microscope and colonies greater
than 50 cells were scored as GM-colony forming units (CFU-GM),
erythroid burst-forming unit (BFU-E), or granulocyte erythroid
megakaryocyte macrophage-colony forming unit (CFU-GEMM).
[0123] LTBMC conditions: The cultures were incubated at 37.degree.
C. in a humidified 5% CO.sub.2/95% air atmosphere and perfused
(medium exchanged) at a rate of 50% daily medium exchange. During
the first week in culture, all cells removed during the daily
medium exchange were centrifuged and returned to the original
wells. After the first week in culture, 50% of the total
nonadherent cells were removed from the cultures on a biweekly
basis during the medium exchange, mononucleated cells counted, and
fresh medium returned to the wells. The remaining five days per
week when the cells were not counted, 50% of the medium was removed
from each of the culture wells and replaced with fresh medium, the
removed medium was centrifuged, the medium decanted from the cell
pellet, and the cells returned to their original wells.
[0124] Statistical analysis: The probability of significant
differences between groups of cultures was determined by comparing
the normalized cumulative cell production values from the rapidly
perfused cultures supplemented with hematopoietic growth factors to
the matched untreated control cultures using a paired t-test.
Statistical significance was taken at the 5% level. There were no
statistical differences between matched rapidly perfused LTBMCs
cultured on tissue culture plastic and type I rat tail collagen at
the 5% level. Therefore, the data for the plastic and collagen
matrix were combined for presentation in this and all other figures
and statistical analysis performed on the combined data.
[0125] Results:
[0126] Kinetics of cell production in rapidly exchanged growth
factor supplemented LTBMCs: As a first test of the hypothesis that
the longevity and production of specific lineages of mature cells
can be influenced by supplementation of HGFS, we established
rapidly exchanged in vitro bone marrow cultures that were
supplemented with IL-3 or Epo. In these cultures, 50% of the medium
was removed daily and replaced with an equal volume of fresh medium
supplemented with IL-3 or Epo. The cells removed were then
centrifuged, the medium decanted and discarded, the cells
resuspended, and the cells returned to the original cultures. IL-3
and Epo individually enhanced the cell productivity of rapidly
exchanged LTBMCs. The cultures containing Epo alone initially had a
high cell production rate due to substantial terminal erythroid
differentiation. However, by week four erythropoiesis had ceased
and the cell production rate had decreased to the level of the
control cultures. IL-3 and Epo induced an average increase in
nonadherent cell production over controls throughout the 18 weeks
of culture of 175% and 173%, respectively.
[0127] Next, combinations of growth factors proved to be more
effective in increasing the nonadherent cell production rate and
the diversity of lineages of cells produced. The highest rate of
cell production was observed for the combination of
IL-3+GM-CSF+Epo. These cultures produced approximately 25% of the
number of cells inoculated biweekly during the first 6 weeks in
culture and had an average 4.8-fold increase in nonadherent cell
production over controls during weeks 2-8. The combination of
IL-3+GM-CSF produced an average 3.5-fold increase in nonadherent
cells as compared to controls through week 8. In separate
experiments, adding neither IL-6 nor G-CSF to the combination of
IL-3+GM-CSF+Epo improved the nonadherent cell production rate, but
instead resulted in cell production rates indistinguishable from
the cultures containing the combination of IL-3+GM-CSF. In all
cases, the stimulatory effect on cell production induced by the
addition of HGFs was maximal between weeks 0 to 8, although cell
production was higher than the controls throughout the culture.
[0128] The combinations of HGFs lead to high absolute numbers of
nonadherent cells produced in rapidly exchanged LTBMCS. The
productivity of the cultures can be shown by comparing the
cumulative number of cells produced over time
(.SIGMA..sup.n.sub.i=1 C.sub.i, C.sub.i being the number of
nonadherent cells collected at time i), relative to the number of
cells inoculated (C.sub.0) by plotting the ratio
(.SIGMA..sup.n.sub.i=1 C.sub.i/C.sub.0) as a function of time. When
this ratio exceeds unity, a culture has produced more cells than
were inoculated and the culture has led to an expansion in cell
number.
[0129] The combination of IL-3+GM-CSF+Epo induced cumulative cell
production that was more than 3-fold greater than the number of
cells inoculated. The cell production rate was the highest during
the first 6 weeks in culture during which time the culture produced
approximately as many cells as were inoculated every two weeks.
This maximum cell production rate was 15% of the estimated in vivo
bone marrow cell production rate where 50% of the myeloid cell mass
is generated daily. The combination of IL-3+GM-CSF resulted in more
than a 2-fold expansion in cell number and at rates comparable to
the combination of IL3+GM-CSF+Epo during weeks 3-7 in culture.
Untreated rapidly exchanged (50% daily medium exchange) and slowly
exchanged (50% medium exchange biweekly) control cultures not
supplemented with HGFs produced approximately 1 and 0.37 times the
number of cells inoculated after 18 weeks, respectively. More
importantly more than half of all cells removed from these
unsupplemented cultures came from the first two samplings,
indicating that many of these cells were from the original inoculum
and that supplementation of the cultures with HGFs are required to
induce significant cycling of progenitor and stem cells.
[0130] Morphologic analysis of nonadherent cells: The addition of
multiple HGFs also increased the variety of myeloid cells produced
in the cultures. The control cultures produced nonadherent cells
that were predominately macrophages after week 3 in the culture.
Production of erythroid cells decreased rapidly with few erythroid
cells detected after week 5. The cultures containing Epo (Epo
alone, IL-3+Epo, and IL-3+GM-CSF+Epo) produced a transient increase
in erythroid cell production, with a high percentage (55-75%) of
nonadherent cells being erythroid through week 3. When
IL-3+Epo.+-.GM-CSF was present, the cultures continued to produce
erythroid cells throughout the 16 weeks in culture with about 5-15%
of the nonadherent cells being typed as erythroid. Thus, in the
presence of IL-3+Epo, erythropoiesis was active throughout.
IL-3.+-.Epo led to a nonadherent cell population that was
predominately (60-70%) late granulocytes (LG) at week 5. The
percentage of LGs steadily declined until it reached about 20% at
week 18. The production of macrophages rose correspondingly. When
GM-CSF was added to IL-3.+-.Epo, the high percentage of LG
persisted through 18 weeks. The combination of IL-3+GM-CSF thus led
to active granulopoiesis for 18 weeks in culture, and the addition
of Epo maintained erythropoiesis as well. Photomicrographs of the
control and IL-3+GM-CSF+Epo supplemented cultures at 5.5 weeks in
culture show the dramatic enhancement in culture density and
variety of cells produced.
[0131] Kinetics of nonadherent Progenitor cell production:
Progenitor cell production increased with the addition of multiple
HGFs. The production of granulocyte macrophage colony forming units
(CFU-GMs) in the untreated controls was prolonged and steady for
over 18 weeks, which is consistent with the earlier results
obtained using rapidly perfused LTBMC without HGF. CFU-GM produced
in the IL-3+GM-CSF and IL-3+Epo.+-.GM-CSF cultures was
approximately 10-fold higher than controls during weeks 3 to 5.
[0132] Erythroid burst forming unit (BFU-E) production in human
LTBMC has been reported to be low and cease quickly (Coutinho et
al, Blood (1990) 75(11): 2118-2129). The rapidly exchanged,
untreated controls exhibited a rapid decrease in BFU-E production
although low levels of BFU-E were produced through 17 weeks in
culture. The addition of Epo alone did not significantly influence
the number of BFU-Es produced. IL-3 alone induced a mild
short-lived stimulation of BFU-E production in weeks 3-5. On the
other hand, in the present cultrues, IL-3 plus either Epo or GM-CSF
induced a 10 to 20-fold elevation in the generation of nonadherent
BFU-E compared to that of controls during weeks 3 to 5 of
culture.
[0133] Discussion:
[0134] It is clear from the data presented that, when combined with
the inventive rapid exchange/perfusion hematopoietic cell culture
conditions (Example 1), supplmentation with judicious combinations
of hematopoietic growth factors leads to increased production of
specific lineages of cells. In this example IL-3+Epo resulted in
sustained red cell production not previously observed in any human
LTBMC. Similarly, IL-3+GM-CSF supplementation resulted in sustained
granulocyte production. Supplementation with different HGFs may
likewise lead to preferential production of different blood cells,
such as platelets, B lymphocytes or T lymphocytes. The critical
feature of the present invention is that the combination of rapid
medium exchange conditions combined with hematopoietic growth
factor supplementation results in essentially physiologic
hematopoiesis, allowing the controlled in vitro generation of blood
cells of the desired lineage or lineages.
[0135] The novelty of the present discovery is evident by
comparison with other recent reports (Coutinho et al, Blood (1990)
75(11): 2118-2129). In these studies evaluating the effect of
supplementation of slowly exchanged LTBMCs with HGFs, although cell
production increases over untreated controls with addition of HGFs,
the increases are smaller and shorter in duration than are reported
here. This discrepancy suggests that physiologic perfusion
stimulates hematopoiesis via a mechanism independent of, and
synergistic with, the effects of IL-3, GM-CSF, or Epo. For example,
as disclosed in the previous parent patent application, increased
serum/medium perfusion rates can induce production of endogenous
hematopoietic growth factors by stromal cells in vitro. Increased
medium perfusion and addition of HGFs may therefore also induce
other HGF production (e.g. kit-ligand) by stimulating hematopoietic
or accessory cells in the cultures. Therefore, increasing the
medium perfusion rate may provide LTMBCs benefits other than just
increasing metabolite and decreasing waste product levels.
Example 3
Use of Rapid Exchange Culture System to Deplete a Bone Marrow Cell
Population of Lymphoid Cells
[0136] The inventors have discovered that the present methods,
including the present culture media conditions, which allow for the
in vitro replication and differentiation of human stem and
hematopoietic progenitor cells can simultaneously promote the
disappearance of specified cells present in the original
composition, notably lymphoid cells such as T-cells and B-cells.
Although the present methods and composition support the
replication and differentiation of human stem and hematopoietic
progenitor cells, human T-cells and B-cells do not proliferate or
maintain viability in these in vitro culture conditions.
[0137] Therefore, in accordance with this embodiment of the
invention, a mixed human hematopoietic mononuclear cell fraction
can be effectively depleted of T-cells and/or B-cells, under
conditions which allow for expansion of stem and progenitor cells.
This method of selective T-cell depletion of human hematopoietic
cell populations has notable therapeutic value as noted above.
[0138] Methods: The Conditions of Culture are identical to those in
Example 2.
[0139] Analysis of B-cells, T-cells, and early Hematoooietic Cells:
At weekly intervals, cells were removed from the cultures and
analyzed by flow cytometry on a FACS Scan (Becton-Dickinson) Cell
Analyzer. B-cells were enumerated with anti-CD19, T-cells with
anti-CD3, and early hematopoietic progenitor cells with anti-CD34,
using fluoresceinated normal mouse Ig as a background control (all
antibodies were from Becton-Dickinson, Inc).
[0140] Results: Human bone marrow cells were cultured as in Example
2, above. Samples were removed prior to the culture and following
7, 14 and 21 days of culture. Aliquots of 2.times.10.sup.5 cells
each were analyzed for the presence of either CD-3, -19, or -34,
and % +cells were determined by subtracting background fluorescence
with a fluoresceinated normal mouse Ig control antibody. As shown
in Table 3 below, T-cells and B-cells were rapidly lost in these
cultures, while primitive hematopoietic progenitor (CD34+) cells
were maintained:
12TABLE 3 Selective Depletion of T and B Lymphocytes from within a
Proliferating Human Bone Marrow Culture. % of Cells in Culture Week
Cell Subset 0 1 2 3 T-cell (CD-3) 20.8 11.2 8.2 0.1 B-cell (CD-19)
5.6 3.0 1.2 0.0 Progenitor cell (CD-34) 0.4 0.7 0.7 0.7
[0141] Thus, the described culture conditions support the survival
and proliferation of hematopoietic progenitor cells, while
simultaneously depleting the culture of T and B lymphocytes. Human
hematopoietic cell populations prepared in this way are directly
applicable to allogeneic bone marrow transplantation, and also to
depletion of virally infected T- and/or B-cells from a
hematopoietic cell population for auto- or
allo-transplantation.
Example 4
Use of Rapidly Exchanged/Perfused Culture System to Deplete a
Hematopoietic Cell Population of Tumor Cells
[0142] In a fashion analagous to that of lymphoid cell depletion,
cancer cells, including T- and B-cell leukemia and lymphoma cells,
and chronic myelogenous leukemia cells, can be depleted from within
a hematopoietic cell population by cultured in a rapid medium
exchanged system. The essential feature of the culture system, that
it allows the survival and proliferation of hematopoietic stem and
progenitor cells while not supporting the survival and
proliferation of other cell types, results in the loss of cancer
cells which are relatively fastidious in their growth and survival
requirements.
[0143] By example, chronic myelogenous leukemia cells (CML) can be
depleted from within a bone marrow cell culture that contains
normal and CML cells, over a culture period of 14-21 days. Under
these conditions, the % of cultured cells that belong to the CML
clone can decrease from 90% or more to under 5%, and in some cases
less than 1%.
[0144] In this way the presently described culture system can be
directly applicable to the purging of cancer cells from within any
hematopoietic cell mass (bone marrow, peripheral blood, cord blood,
fetal blood) for autologous or allogeneic bone marrow
transplantation, and can be applied directly to the treatment of
cancer patients.
[0145] The exact time of culture required to achieve substantial
depletion of the malignant cells may depend on the exact type and
the number of malignant cells present. However, the time required
to achieve a culture substantially free of any particular type of
malignant cell may be easily determined by one of skill in the art
by using the present method in conjunction with any conventional
assay for the type of malignant cell in question.
Example 5
Use of Rapidly Exchanged/Perfused System to Assay the Influence of
Specified Molecules on Hematopoiesis
[0146] We now demonstrate how the culture system described in
Example 2 can be used to assay the effects of any individual or
combination of additional specified substances. In this embodiment,
this system provides a method for assaying the affect of a
substance or substances on a hematopoietic cell culture. In
accordance with this embodiment, one adds to a cell culture carried
out in accordance with the invention at least one substance
suspected of having an affect, which may be either beneficial or
detrimental, on the cell culture. One then compares the cell
culture profile obtained in the absence of the substance being
tested to the cell culture profile obtained in the presence of the
substance. This embodiment may be used in accordance with the
various embodiments used in the present invention to ascertain the
particular affect of a suspected substance on a human stem or
hematopoietic cell system. As examples, Interleukin 6 (IL-6) and
Granulocyte colony stimulating factor (G-CSF), are two
hematopoietic growth factors with well described hematopoietic
activities on already-developed progenitor cells. The present assay
system provides an improved approach to analyzing the effects of
these HGFs along with those of IL-3 and GM-CSF, both on progenitor
cells and on primitive hematopoietic progenitor cells.
[0147] Materials and Methods:
[0148] Cells, hematopoietic progenitor cell assays, LTBMCs, and
statistical analyses were as in Example 1.
[0149] Hematopoietic growth factors: IL-3, 1 ng/ml; GM-CSF, 1 ng/ml
of GM-CSF; Epo, 0.1 U/ml; IL-6, 10 U/ml (Collaborative Research
Inc.), and G-CSF, 0.1 ng/ml (Amgen Biologicals).
[0150] Results:
[0151] Kinetics of nonadherent cell production: Supplementation of
IL-6 or G-CSF to the combination of IL-3+GM-CSF+Epo reduced the
number of granulocytes produced. IL-3+GM-CSF+Epo with IL-6 or G-CSF
resulted in an approximate 2-fold decrease in late granulocyte
production similar to the reduction seen in early granulocyte
production. Interestingly, macrophage production through 6.5 weeks
in culture was not significantly affected by any combination of
growth factor supplementation in the LTBMCs. Cumulative macrophage
production varied between 0.84 and 1.4.times.10.sup.6 cells per
culture for the untreated and HGF supplemented cultures (Table
4).
13TABLE 4 Cumulative cell production by cell lineage in growth
factor supplemented LTBMCs over the first 6.5 weeks of culture.
Early Late Eryth- Granu- Granu- Macro- roid locytes locytes ratio
phages Culture (E) (EG) (LG) (LG/EG) (Mo) control 0.41 0.76 1.6 2
0.84 IL-3 0.14 3.0 3.1 1 2.9 GM-CSF 0.11 2.7 6.8 2.5 2.1 IL-3 +
GM-CSF 0.04 4.5 11.9 2.5 1.2 IL-3 + GM-CSF + 3.40 4.7 11.8 2.5 1.4
Epo IL-3 + GM-CSF + 3.50 3.2 6.9 2 1.4 Epo + IL-6 IL-3 + GM-CSF +
3.10 3.4 6.9 2 1.6 Epo + G-CSF
[0152] The data in Table 4 clearly shows the effects of the growth
factors on the kinetics of lineage production in the cultures. For
instance, by comparing the cumulative production of erythroid cells
in the IL-3+GM-CSF and IL-3+GM-CSF+Epo supplemented cultures (lines
4 and 5 in Table 4) one can clearly assay for the effects of Epo on
the reconstitution of erythropoiesis in vitro. Both of these
cultures produce the same number of granulocytes and macrophages
but they differ vastly in their production of erythroid cells. The
culture with Epo produced 3.4 million cells while the one without
Epo produces undetectable amounts of erythroid cells. Similarly, by
comparing lines 5 and 6 in table 4 one can evaluate the influence
of IL-6 on lineage productivities. IL-6 does not influence the
production of erythroid cells but diminishes the number of early
granulocytes produced and their maturation to late
granulocytes.
[0153] In an analogous fashion, one can use this in vitro tissue
system as an assay system for the effects of synthetic and/or
natural substances on lineage reconstitution or depletion as well
as the survival (or purging) of malignant cells in (from)
populations of bone marrow cells.
[0154] Progenitor cell production: Progenitor cell production was
dramatically influenced by addition of HGFs to LTBMCs. Granulocyte
macrophage progenitor cell (CFU-GM) production was also affected by
HGF supplementation of the LTBMCs. Individually, IL-3 or GM-CSF
produced only a slight increase in total CFU-GM (Table 5) whereas
IL-3+GM-CSF+Epo induced approximately a 1.7-fold increase in total
CFU-GM production. IL-3+GM-CSF+Epo with IL-6 or G-CSF induced a 2.4
and 3.1-fold increase in the number of CFU-GM removed from culture,
respectively. These results suggest that IL-3 and GM-CSF act
synergistically to recruit early myeloid cells into the granulocyte
and macrophage lineages and that Epo has little effect on CFU-GM
production. Interestingly, IL-6 and more significantly G-CSF when
combined with IL-3+GM-CSF+Epo, had a large positive effect on the
number of CFU-GM when removed from culture whereas they had little
effect in the erythroid lineages as shown by BFU-E production
suggesting a more restricted action of these HGFs.
[0155] CFU-GEMM production was significantly influenced by HGF
supplementation. Individual supplementation of the LTBMCs with
either IL-3 or GM-CSF alone, increased the number of CFU-GEMM
removed from culture 1.8 and 1.6 fold, respectively. However,
combinations of CSFs induced a much larger increase in CFU-GEMM
production than did either IL-3 or GM-CSF alone. Supplementation
with IL-3+GM-CSF and IL-3+GM-CSF+Epo induced a 3.5 and 10.3-fold
increase in the total number of CFU-GEMM removed, suggesting that
Epo plays either a direct or indirect role in early hematopoietic
events. The addition of IL-6 or G-CSF to the combination of
IL-3+GM-CSF+Epo did not affect CFU-GEMM production above
IL-3+GM-CSF+Epo alone (Table 5).
14TABLE 5 The cumulative number of progenitor cells removed from
rapidly perfused HGF supplemented human long- term bone marrow
cultures. culture BFU-E CFU-GM CFU-GEMM control 700 1900 50 IL-3
1900 2100 90 GM-CSF 800 2200 80 IL-3 + GM-CSF 2500 3200 200 IL-3 +
GM-CSF + Epo 3000 3200 500 IL-3 + GM-CSF + Epo + IL-6 3200 4600 500
IL-3 + GM-CSF + Epo .+-. IL-6 3200 5900 500 or G-CSF
[0156] Production of Progenitor Cells from Stem Cells. Although the
most primitive hematopoietic cells that can be directly assayed are
the progenitor cells; CFU-GEMM, CFU-GM, and BFU-E, the presence and
activity or earlier stem cells can be inferred by the continued
production of progenitor cells. Thus, progenitor cell production is
an indirect measure of primitive stem cell activity. One can assay
stem cells, therefore, by analyzing the kinetics of the progenitor
cell pool. The four process rates that determine the progenitor
cell pool size are:
[0157] V.sub.1: The rate of production of progenitor cells by more
undifferentiated stem cells;
[0158] V.sub.2: The rate of loss of progenitor cells through the
act of sampling of nonadherent cells from the culture;
[0159] V.sub.3: The rate of loss of progenitor cells by their
differentiation to mature cells of a particular lineage;
[0160] V.sub.4: The rate of death of progenitor cells. The initial
number, C.sub.0, of each progenitor cell species can be determined
by assay at culture initiation.
[0161] The question that we wish to answer is; Are stem cells being
stimulated to produced progenitor cells--i.e. is V.sub.1 greater
than zero? Cell death, V.sub.4, cannot be assessed. V.sub.2
however, can be measured by assaying the nonadherent cells removed
from culture, and V.sub.3 can be estimated from nonadherent cell
production data. A conservative estimate of the rate of progenitor
cell differentiation, V.sub.3, can be determined by assuming that
each progenitor cell has 10 divisions remaining to become a
terminally differentiated cell. Therefore, each progenitor cell is
equivalent to 2.sup.10 (1024) mature cells. From the total number
of mature cells produced of a specific lineage, the number of
progenitor cells (BFU-E and CFU-GM) that differentiated can be
back-calculated. Therefore, if the removal and differentiation of
progenitor cells (V.sub.2+V.sub.3) exceeds the numbers of
progenitor cells inoculated, C.sub.o, then progenitor cells must
have been produced, and V.sub.1 is greater than zero.
[0162] We applied these calculation to two measured progenitor cell
pools, CFU-GM and BFU-E. Tables 6 and 7 show that only in the
cultures supplemented with IL-3+GM-CSF+Epo, IL-3+GM-CSF+Epo+IL-6,
or IL-3+GM-CSF+Epo+G-CSF did the total number of BFU-E that could
be measured (those that were removed and those that differentiated)
in culture exceed those inoculated. In these cultures, the removal
of BFU-E was approximately equivalent to those that differentiated
(Table 5). Similar calculations for CFU-GM show that production of
CFU-GM (Table 6) was greater than zero (V.sub.1>0) in all
cultures. However, IL-3+GM-CSF or IL-3+GM-CSF+Epo supplementation
induced a 5.1-fold increase in CFU-GM production over the control
cultures which itself was 8.4-fold greater than the inoculum. Thus
the combination of IL-3 and GM-CSF stimulated the differentiation
of stem cells to progenitor cells.
15TABLE 6 The cumulative production of BFU-E removed and
differentiated in growth factor supplemented rapidly perfused human
long-term bone marrow cultures. Number of Number of erythroid
Estimated BFU-E cells number of Total removed removed BFU-E that
number of (plated) (.times. 10.sup.-6) differentiated BFU-E Culture
(V.sub.2) (V.sub.3) (V.sub.3) (V.sub.1) Inoculum (2900) (0) (0)
(2900) control 700 0.4 400 1100 IL-3 1900 0.1 100 2000 GM-CSF 800
0.1 100 900 IL-3 + GM-CSF 2500 0.0 0 2500 IL-3 + GM-CSF + 3000 3.4
3400 6400 Epo IL-3 + GM-CSF + 3200 3.5 3500 6700 Epo + IL-6 IL-3 +
GM-CSF + 3200 3.1 3100 6300 Epo + G-CSF Every fourth biweekly
sampling from the culture was assayed for BFU-E and the non-assayed
values were estimated by linear interpolation between two known
data points (V.sub.2).
[0163] Discussion: The example extends our understanding and use of
the prolific rapidly perfused HGF supplemented human LTBMCs
described in Examples 1 and 2. We have outlined the kinetics of
lineage reconstitution in this culture system and have shown how
HGF supplementation influences mature and progenitor cell
production.
[0164] Although there are many uses of this system for the analysis
of the effects of added substances, for this example we focus on
the analysis of the effects of IL-6 and G-CSF versus
GM-CSF+IL-3+Epo on progenitor cell production from stem cells. A
population balance analysis of the progenitor and nonadherent cell
production data showed that progenitor cells can be produced in HGF
supplemented rapidly perfused LTBMCs. This analysis showed that all
rapidly perfused cultures produced CFU-GM although the number of
CFU-GM produced is strongly growth factor dependent. BFU-E, on the
other hand, were only produced in the cultures supplemented with
(IL-3+GM-CSF+Epo)+IL-6 or +G-CSF. Analysis of CFU-GM production
alone would indicate that the cultures supplemented with
IL-3+GM-CSF+Epo and either IL-6 or G-CSF stimulated CFU-GM
production more than the combination of IL-3+GM-CSF+Epo alone.
However, a population balance of the total CFU-GM production (Table
7), shows that IL-6 and G-CSF did not induce increased CFU-GM
production over the combination of IL-3+GM-CSF+Epo. Instead, this
analysis suggests that the effect IL-6 and G-CSF on the CFU-GM pool
was to change the cells from a more adherent compartment to a less
adherent compartment, making the cells available for removal during
the bi-weekly sampling. This analysis shows that both
differentiation and sampling must be taken into account to better
understand the effect of growth factors on progenitor cell
production.
[0165] In summary, this example demonstrates how the rapid medium
exchange/perfusion hematopoietic cultures allow for the precise
analysis of the hematopoietic activity of added molecules,
particularly in regard to their effects on hematopoietic stem
cells. This system can be used to analyze the hematopoietic
activity of any desired soluble substance or substances.
16TABLE 7 The cumulative production of CFU-GM removed and
differentiated in growth factor supplemented rapidly perfused human
long-term bone marrow cultures. Number of Number of GM Estimated
CFU-GM cells number of Total removed removed CFU-GM that number of
(plated) (.times. 10.sup.-6) differentiated CFU-GM Culture
(V.sub.2) (V.sub.3) (V.sub.3) (V.sub.1) Inoculum (2500) (0) (0)
(2900) control 1900 3.2 3200 4100 IL-3 2100 9.0 9000 11,100 GM-CSF
2200 11.7 11,700 13,900 IL-3 + GM-CSF 3200 17.6 17,600 20,800 IL-3
+ GM-CSF + 3200 17.9 17,000 20,200 Epo IL-3 + GM-CSF + 4600 11.5
11,500 16,100 Epo + IL-6 IL-3 + GM-CSF + 5900 11.9 11,900 17,800
Epo + G-CSF Every fourth biweekly sampling from the cultures was
assayed for CFU-GM and the non assayed values were estimated by
linear interpolation between two known data paints (V.sub.2).
Functioning, Reconstructed In Vitro Bone Marrow Culture
[0166] Process Flowsheet:
[0167] A process for the carrying out the present invention will
now be described in relation to a preferred embodiment. The liquid
medium may be pumped with a syringe pump that may be located in a
refrigerator adjacent to an incubator, maintained at a temperature
sufficient to sustain hematopoiesis. The fresh medium may be kept
at 4.degree. C. preventing decay of chemically unstable medium
components such as glutamine and growth factors. The medium may be
fed through a PharMed tubing (Norphrene based tubing). This tubing
may have a "slack" so that the syringe pump can be moved to a
laminar flow hood where the syringes can be replaced in a sterile
environment. The extra tubing may be kept in the refrigerator so
that only a short tube segment is at room or at incubator
temperature. This arrangement is important since liquid residence
time in the tube can be on the order of days (depending of the flow
rate used).
[0168] The gas may come from either a cylinder containing premixed
gases (a mixture of O.sub.2, N.sub.2 and CO.sub.2) or may be simply
taken from the inside of the incubator (a mixture of air and
CO.sub.2). The flowrate and composition of the gas stream may thus
be easily controlled. The gas may be pumped with an aquarium pump
through a sandstone in a 100 ml medium flask to give relative
humidities as close to 100% as possible. The gas line may contain a
sterile filter.
[0169] Th spent medium may be collected in a 100 ml medium bottle.
Samples may be taken from it for analysis of medium components. The
sets of chambers may be kept in an CO.sub.2 incubator with a
humidification system.
[0170] Component Description:
[0171] Perfusion Chamber. The perfusion chamber may be made from
two specially machined pieces of a polycarbonate slab, the chamber
top and bottom. Between the top and bottom piece two identical 1/4
inch silicone rubber gaskets may be placed.
[0172] When the chamber is assembled, a membrane may be placed
between the two silicone gaskets which in turn may be placed
between the top and bottom piece and four bolts may be used to hold
the chamber together. The difference between the top and bottom
piece is the number of ports provided. The bottom piece may have
two ports for gas inlet and outlet, whereas the top piece may have
three ports, a medium inlet and outlet and a sample port, placed in
the middle of the top piece. The outlet port may be constructed so
that an angle is formed relative to the horizontal position to
provide gravity induced settling for any non-adherent cells that
might be floating about the chamber. The geometry of the hole in
the silicone gasket may be circular, but an elliptical shape with
the inlet and outlet ports placed in focal points of the ellipse
may provide a better fluid distribution.
[0173] To provide for gas mass transfer, cell/extra-cellular matrix
attachment and to prevent water leakage a two membrane system may
be used. The lower membrane may be a gas exchange membrane, such as
silicone, teflon, mylar, etc. The membrane is preferably
hydrophobic to prevent loss of water and is permeable to gases.
Furthermore, the gas exchange membrane can provide a mechanical
support for the second membrane. The second membrane is for cell
attachment and growth and may be, e.g., an inorganic ceramic based
membrane. It can be coated with extra-cellular protein. We have
used the PepTite-2000.RTM. RGD based adhesion protein (a product of
Telios Pharmaceutical, Inc., San Diego, Calif.) successfully for
this purpose. Further, a highly desirable property of a ceramic
inorganic membrane is that it becomes transparent upon hydration
thus making microscopic observation of the cells possible. The
second membrane may serve as a surface for the attachment of the
adherent cells.
[0174] The only difficulty encountered with the two membrane system
arises from the fact that the thermal expansion coefficient for the
inorganic membrane exceeds that of other chamber components. Thus,
during autoclaving the bolts that hold the chamber together cannot
be tightened. Even with loose bolts, we have experienced that about
20% of the chambers have a cracked inorganic membrane after
autoclaving.
[0175] The tubing for medium and gases may be connected to the top
and bottom pieces using polypropylene fittings and Luer Locks
rings. By employing silicone O-rings, good seals are formed and no
leakage problems have occurred.
[0176] The perfusion chamber can be assembled in other
configurations. Two alternatives are:
[0177] (1) An inverted configuration. The configuration described
above is inverted, and the cells grow on the bottom plate. In this
case, only the gas exchange membrane is required; and
[0178] (2) A three-chamber design. Gas is circulated through a top
compartment which is separated from a central compartment by a gas
exchange membrane. The cells are in the central compartment which
is separated from a bottom compartment by a cell growth membrane.
The central compartment is stagnant, while the bottom compartment
is continuously perfused.
[0179] Auxiliary Components. The syringe pump should carry up to
ten syringes (for a set of ten parallel chambers) of the
appropriate size. Currently we are using a flowrate of 2 ml/day and
10 ml syringes, requiring syringe change every 5 days. The pump
should be accurate, reliable, and be able to operate at 4.degree.
C. over extended periods of time. We have used a Harvard `22`
syringe pump with a multiple syringe holder. These pumps have
proved satisfactory, although twice they have broken down after
long periods of use at 4.degree. C.
[0180] The air may be pumped with an aquarium pump. Medium bottles
may be used for the spent medium and for the humidification of the
incoming gas stream. Each component is subject to modifcation. For
the spent medium bottle, e.g., one can add a septum that would
allow for sterile sampling or depletion of the spend medium. A tall
and narrow bottle might be preferable for humidifying a stream of
premixed gases since they are at very low relative humidity. When
air from the incubator is used this precaution is not necessary
since the incubator air is at high relative humidity.
[0181] Operating Procedures:
[0182] Starting up the Perfusion Chambers:
[0183] Cells. The cells are treated prior to inoculation in the
same fashion as they are prepared for Dexter cultures. After
aspiration from a donor, mononuclear cells are separated on a
discontinuous density gradient (Ficoll) and then washed several
times in the culture medium. This procedure typically takes about
half a day.
[0184] Medium. The medium used is the standard Dexter medium, 10%
horse serum, 10% fetal calf serum, 10.sup.-6 M hydrocortisone and
IMDM. In addition, hematopoietic growth factors are added.
Typically, Il-3, GM-CSF and Epo are used as previously described.
The search continues for the optimal combination of added
hematopoietic factors.
[0185] Perfusion Chambers. The preparation of the perfusion
chambers starts one day prior to inoculation. Assembly of a set of
6-10 perfusion chambers takes about 6 to 8 hours. This involves
sizing/cutting tubing, putting fittings into the chamber, preparing
the medium bottles, etc. At the end of the day the full chamber
assembly (less the tubing and attachments for the gas exchange) is
autoclaved without medium (all components are autoclaved). The set
of chambers is then typically stored in a hood overnight. The
following day the full set of components is assembled in the hood,
the medium introduced, any adhesion protein applied, cells
inoculated, the chambers placed in the incubator, the syringes
loaded into the pump and stored in the refrigerator. If cell
preparation is included, these steps take another day. Thus, the
chambers are running at the end of the second say. The perfusion
typically begins after the cells have settled in the chamber for 12
to 24 hours.
[0186] Running the Perfusion Chambers:
[0187] Once the chambers are set up they are easy to maintain; the
syringes need to be replaced at a fixed interval and non-adherent
cells collected.
[0188] Replacing Syringes. The syringes are typically replaced on a
fixed schedule. For instance, during the initial runs with the
chambers, 10 ml syringes were used at a flow rate of 2 mls per day.
Syringes thus were replaced every 5th day. The syringe pump is
moved from the incubator to the hood where the syringes are
replaced in a sterile environment. This transfer of the pump is
allowed by the "slack" in the medium inlet line as described above.
We have yet to experience contamination problems during this
procedure.
[0189] Microscopic observation. The top and bottom of the perfusion
chamber and the teflon membrane are transparent. The inorganic
membrane becomes transparent once it is hydrated and thus during
operation one can observe the cells in the chamber through a
microscope. To do so one needs a long distance objective.
[0190] Sampling cells. Sampling the cells from the chambers can be
achieved using one of two methods. Firstly, we let cells settle by
gravity inversion for two hours and then we replace 2 mls in the
chamber by pushing liquid through the inlet port and collecting it
from the outlet line. Secondly, we have pulled directly through the
sampling port 2 mls leaving air space in the chamber that then
disappears within a day due to the incoming medium. The second
method is more invasive and yields a higher number of cells
(approximately two to four fold).
[0191] The cell sampling takes place in the laminar flow hood. The
set of chambers is moved from the incubator to the hood and the
length of the inlet medium line allows for this transfer. Since the
solubility of oxygen is higher at lower temperatures, the cooling
to room temperature often results in the formation of bubbles in
the chambers (the bubbles are often located between the two
membranes) once they are returned to the incubator. Such bubbles
disappear after readjustment to 37.degree. C.
[0192] The adherent cells can be removed in a similar fashion after
treatment with trypsinization.
[0193] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that, within the scope of the
appended claims, the invention may be practiced otherwise than as
specifically described herein.
* * * * *