CA1310926C - Process for replicating bone marrow and other tissues in vitro and using the same - Google Patents
Process for replicating bone marrow and other tissues in vitro and using the sameInfo
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- CA1310926C CA1310926C CA000534951A CA534951A CA1310926C CA 1310926 C CA1310926 C CA 1310926C CA 000534951 A CA000534951 A CA 000534951A CA 534951 A CA534951 A CA 534951A CA 1310926 C CA1310926 C CA 1310926C
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Abstract
ABSTRACT
A process for treating a person whose bone marrow has been destroyed or has lost its functional ability, which includes the steps of obtaining bone marrow from a donor, replicating the bone marrow cells in vitro, and then reinfusing the replicated marrow cells at a later date. The bone marrows may be cryopreserved before and/or after the replication step. The in vitro bone marrow replication system may also be used to monitor a patient's condition or the cytotoxicity of various agents.
Three-dimensional cell culture systems for the growth of at least two layers of cells, for cell types in addition to bone marrow cells, may also be established using a three-dimensional support as a framework.
A process for treating a person whose bone marrow has been destroyed or has lost its functional ability, which includes the steps of obtaining bone marrow from a donor, replicating the bone marrow cells in vitro, and then reinfusing the replicated marrow cells at a later date. The bone marrows may be cryopreserved before and/or after the replication step. The in vitro bone marrow replication system may also be used to monitor a patient's condition or the cytotoxicity of various agents.
Three-dimensional cell culture systems for the growth of at least two layers of cells, for cell types in addition to bone marrow cells, may also be established using a three-dimensional support as a framework.
Description
PROCE5S FOR REPLICATIN~ BONE MARROW AND OTHER
TISSUES IN VITRO AND USING THE SAME
Processes for transplanting bone marrow are known.
Generally, bone marrow transplants are performed on patients who suffer from a disease, such as cancer, which destroys healthy bone marrow cells or depresses their functional ability. In addition, treatments such as chemotherapy or radiation therapy adversely affect the bone marrow even in cases where the bone marrow has not been directly affected by the disease being treated. Rnown methods of bone marrow transplantation suffer from a number of disadvantages. One major cause of bone marrow transplant rejection is the graft versus host reaction which occurs when the bone marrow removed from one person is transplanted into another person.
Another major cause o~ bone marrow transplant failure is vasoocclusive disease resulting from the formation of marrow emboli. Procedures for the removal and storage of a persons' marrow prior to combined chemotherapy and radiation and reinfusion of that marrow currently exist (i.e., autologous transplant). However, the patient often suffers from the recurrence of the disease even if engraftment occurs since the marrow was already diseased when f irst removed.
Therefore, it is the object of the present invention to provide a process for treating diseases that destroy healthy bone marrow cells or depress their functional or replicative ability which process does not suffer from the known disadvantages of current bone marrow transplantation techniques.
Another object of the present invention is to provide a process for treating diseases by bone marrow transplantation which process does not produce a graft versus host reaction.
A further object of the present invention is -
TISSUES IN VITRO AND USING THE SAME
Processes for transplanting bone marrow are known.
Generally, bone marrow transplants are performed on patients who suffer from a disease, such as cancer, which destroys healthy bone marrow cells or depresses their functional ability. In addition, treatments such as chemotherapy or radiation therapy adversely affect the bone marrow even in cases where the bone marrow has not been directly affected by the disease being treated. Rnown methods of bone marrow transplantation suffer from a number of disadvantages. One major cause of bone marrow transplant rejection is the graft versus host reaction which occurs when the bone marrow removed from one person is transplanted into another person.
Another major cause o~ bone marrow transplant failure is vasoocclusive disease resulting from the formation of marrow emboli. Procedures for the removal and storage of a persons' marrow prior to combined chemotherapy and radiation and reinfusion of that marrow currently exist (i.e., autologous transplant). However, the patient often suffers from the recurrence of the disease even if engraftment occurs since the marrow was already diseased when f irst removed.
Therefore, it is the object of the present invention to provide a process for treating diseases that destroy healthy bone marrow cells or depress their functional or replicative ability which process does not suffer from the known disadvantages of current bone marrow transplantation techniques.
Another object of the present invention is to provide a process for treating diseases by bone marrow transplantation which process does not produce a graft versus host reaction.
A further object of the present invention is -
-2- 1 31 0926 to provide a process for treating diseases by bone marrow transplantation which does not cause the formation of marrow emboli.
Yet another object of the present invention is to provide a means for treating diseases or conditions that destroy hçalthy bone marrow cells by aspirating bone marrow from a patient, replicating the bone marrow cells in vitro and then reinfusing the replicated marrow cells into the patient.
A still further object of the present invention is to provide a process for increasing the functional ability of a bone marrow which has been adversely affected by chemotherapy and/or radiation therapy.
Still another object of the present invention is to provide a process for the treatment of hematological malignancies and other neoplasias which metastasize to the bone marrow.
Additional objects and advantages of the present invention will be apparent to those skilled in the art by reference to the following detailed description.
The invention may be better understood by reference to the single figure of the following drawings showing a scanning electron photomicrograph at a magnification of 1. 65 thousand times showing a meshwork with bone marrow stromal cells growing thereon.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a process for treating a person whose bone marrow has been destroyed or lost its functional ability. The process comprises the steps of:
I. obtaining a bone marrow sample from a ` donor; and then II. replicating the bone marrow sample in vitro to produce a replicated bone marrow _3_ 1310q26 containing hematopoietic stem cells having marrow repopulating activity, a portion of which may be cryopreserved for later use;
and then III. infusing the replicated bone marrow containing hematopoietic stem cells into the person to restore hematopoiesis in the person.
According to the present invention, there is provided a process for treating diseases or conditions which have destroyed healthy bone marrow cells or have depressed their functional ability. The process of the present invention is especially effective in the treatment of hematological malignancies and other neoplasias which metastasize to the bone marrow.
The process of the present invention comprises the steps of: aspirating a small amount of bone marrow from a healthy patient; cryopreserving said cells; replicating the bone marrow cells in vitro to increase the number of bone marrow cells to a number sufficient for autologous reinfusion, which is greater than the number originally removed from the patient; and then reinfusing the bone marrow cells into the patient. According to one embodiment of the present invention, the bone marrow cells are cryopreserved or frozen immediately after aspiration from the patient and are subsequently thawed and replicated. According to another embodiment of the present invention, an allogeneic bone marrow transplant is performed by aspirating bone marrow from one person, replicating the bone marrow cells in vitro and then reinfusing the replicated marrow cells into another person.
The in vitro bone marrow replication system of the present invention also provides a means to monitor a patient in regard to those conditions that affect the bone marrow.
For example, a small sample of bone marrow is aspirated from _4~ U q 26 a treated cancer patient and replicated in the ln vitro system of the present invention, in order to check for a recurrence or metastasis of the original malignancy.
The in vitro bone marrow replication system is also adaptable for cytotoxicity testing of pharmaceuticals, food additives, health products, anti-neoplastic agents, and carcinogens. A proportional, scaled-down dose of the agent to be tested is added ~o the ln vitro bone marrow system of the invention and its effect on various cell types noted.
Cell types other than bone marrow cells are substituted to test a particular agent, as required.
The invention also describes a three-dimensional support for growing cells in culture. The growth of cells in the presence of this support may be further enhanced by adding proteins, glycoproteins, glycosaminoglycans, a cellular matrix, and other materials to the support itself or by coating the support with these materials. The use of a three-dimensional support allows the cells to grow in multiple layers, thus creating the three-dimensional, cell culture system of the present invention. The three dimensional support can be used to create three-dimensional, cell culture systems for bone marrow, skin, liver, and many other cell types and tissues. In one embodiment of this invention, the three-dimensional cell culture system may be transferred onto or into a living orgamism.
The three-dimensional support is preferably in the form of a mesh. Prepared supports may be preserved for later use in a three-dimensional, physiologic, cell culture system.
In an embodiment for growing hematopoietic bone marrow cells, the mesh i5 prepared by growing a layer of stromal marrow cells on the mesh until the stromal marrow cells reach subconfluency.
BRIEF DESCRIPTION OF THE DRAWING
Referring now to the single figure of the drawings, ~5~ 1 3 1 0~6 there is shown a meshwork 10 useful in the present invention.
The meshwork 10 contains warp threads 12 and woof threads 13.
As shown in the figure, stromal cells 14 can be seen growing on the threads 12, 13.
DETAILED DESCRIPTION OF THE INVENTION
Replication and Cyropreservation of Bone Marrow for Use in Bone Marrow Transplantations The present invention is directed to a process for treating diseases or conditions which destroy healthy bone marrow cells or depress their functional ability. The process is effective especially in the treatment of hematological malignancies and other neoplasias which metastasize to the bone marrow. The process of this invention is also effective in treating patients whose bone marrow has been adversely affected by chemotherapy and/or radiation therapy necessitated by a disease which does not directly affect the bone marrow. The process of the present invention further provides a means for removing and preserving bone marrow cells from a healthy patient and then replicating and reinfusing the bone marrow cells should the patient develop an illness which either destroys the bone marrow directly or whose treatment adversely affects the marrow.
In accordance with the process of the present invention, a small amount (10-15 cc bone marrow/peripheral blood suspension) is aspirated from the iliac crest of a donor. The results of the process are optimal if: 1. The individual is under 40 years of age at the time his/her marrow is cryopreserved, 2. the patient is disease-free.
This procedure may prove beneficial to certain patients with metastatic disease or hematological malignancies if "purging"
of malignant cells by physical or chemotherapeutic means can be performed prior to culturing. At present, these methods are most efficient when small numbers of cells are used, a fact which might lend itself well to the following procedure.
Methods of aspirating bone marrow from a donor are well known in the art. Examples of apparatus and processes for aspirating bone marrow from a donor can be found in U.S.
patents 4,481,946 and 4,486,188.
The bone marrow removed from the donor is then replicated or preserved for replication at a later date. If the bone marrow is to be preserved, the bone marrow can be incrementally frozen using computerized cryotechnological equipment. Fresh bone marrow/blood suspension is aliquoted in equal volumes into sterile Nunc tubes and placed in a beaker of crushed ice until the cryopreservation chamber is brought to a similar temperature (4C). Immediately prior to specimen insertion into the chamber, a solution is added to each Nunc tube using sterile technique, so that the cryoprotectants, dimethylsulfoxide and glycerol, will be in final concentrations of 7% and 5% respectively. The freezing program is initiated immediately after introduction of the specimen. Freezing program number 1 on the CryoMed Model Number 1010 controller is used.
Using this technique, the cellular viability after freezing and rapid thawing in an 80C water bath exceeds 90%
via the trypan blue exclusion method. In addition, greater than 80% of the original colony forming unit culture (CFU-C) may be recovered after freezing. Examples of systems for freezing bone marrow and biological substances in accordance with a precalculated temperature and time curve are disclosed in U.S. patents 4,107,937 and 4,117,881. Preferably, the bone marrow cells are stored in the liquid phase of liquid nitrogen at a temperature of -196C at which temperature all cellular metabolic activity has ceased.
After thawing, the bone marrow cells is reinfused into an individual or is replicated, and then reinfused.
^~ ~ * Trade Mark _7_ 1 3 1 Oq26 The process of the present invention has several advantages to a patient in need of a bone marrow transplant.
If the patient is receiving his or her own cells, this is called an autologous transplant. Such a transplant has little likelihood of rejection. Autologous transplants eliminate a ~ajor cause of bone marrow transplant rejection, that is, the graft vs. host reaction. Further, when grown in culture, hematopoietic cells may be easily separated by physical manipulation and/or enzyme treatment. This diminishes the risk of vasoocclusive disease resulting from marrow emboli. In addition, the process of the present invention allows more aggressive treatment of neoplastic disorders with chemotherapeutic agents and radiation.
Presently, the extent of these treatments is often limited by bone marrow toxicity.
In Vitro Bone Marrow Replication System The process of the present invention further comprises the step of replicating the bone marrow cells in vitro, in a system comparable to physiologic conditions.
Moreover, the bone marrow cells replicated in this system include all of the cells present in normal bone marrow, assuming all cell types were present in the original bone marrow inoculum.
The bone marrow cells, either obtained directly from the donor or retrieved from cryopreservative storage, are first separated from their reticulum by physical means.
The bone marrow cells are then grown in co-cultures with stromal components of normal marrow, which includes fibroblasts, macrophages, reticular cells, and adipocytes, on a three-dimensional support. Factors derived from media of splenic and/or hepatic (liver) macrophage cultures or from subsets of stromal cells may also be added to the culture~
Although marrow cells are capable of limited growth when cultured alone, long term growth of these cultures is ;, -8- 1 31 Oq26 possible only if stromal cells or their secretory products are present. See Long-Term sone Marrow Culture, D.G. Wright & J.S. Greenberger, eds., A.R. Liss, New York, (1984).
The present invention seeks to maximize the proliferation of multipotential hematopoietic stem cells which have the capability of repopulating bone marrow when the bone marrow has been destroyed by intrinsically or environmentally-mediated disease or by the treatment of such disease with chemotherapy and/or radiation. Stem cells which have marrow repopulating activity (MRA) have been shown to persist and replicate in the long term bone marrow cultures.
However, stem cells, hemopoietic progenitor cells, hemopoietic precusor cells all replicate and proliferate in the system of the present invention. Furthermore, differentiation can proceed in this system in a physiologic manner. For example, erythroid, myeloid, lymphoid, macrophagic, and megakaryocytic colonies, can continuously arise in the same culture system using the systems as taught by the present invention.
According to a preferred embodiment of the present invention, hematopoietic stem cell growth is accomplished as follows:
1. establishment of the stromal support layer Marrow suspensions are centrifuged at 3000 x g for 20 minutes and the white base of cells containing macrophages, fibroblasts, adipocytes, mononuclear blood cells, reticular cells, endothelial cells and other resident cells is removed. The cells are suspended in a medium called Roswell Park Memorial Institute medium number 1640 or simply "RPMI 1640~. The RPMI 1640 is purchased from GIBCO
Incorporated of Grand Island, New York, USA. The RPMI 1640 supplemented with 10% fetal bovine serum, 10% horse serum, hydrocortisone hemisuccinate, and appropriate antibiotics.
106 of these cells are plated onto sterile nylon mesh from the Tetko Corp. of New York, New York, USA in a petri dish. This mesh has been pre-cut to conform to the inside dimensions of a 25mm2 plastic culture flasks. The mesh has a sieve area of 400~m2 and a fiber diameter of lOO~m-The inoculated mesh is then wound and placed intothe culture flask containing 5 milliliters of media as previously described. The mesh unwinds inside the culture flask and completely covers the bottom of the flask. The cultures are grown at 37C in 5% C02 in ambient air at a relative humidity in excess of 90%. Stromal cells which are predominantly fibroblasts first grow along and completely encircle all of the nylon fibers before beginning to grow into the mesh openings. This process takes approximately 14 to 18 days. The degree of subconfluency of the stromal cells, should be consistent with that seen in the figure of the drawing, prior to the inoculation of hematopoietic cells.
Suspended stromal cells can be cryopreserved using the same technigue as previously described for bone marrow cells. For cryopreservation of sub-confluent cells on the mesh, the nylon mesh must be rolled and inserted into the Nunc tube containing RPMI 1640 medium with the cryoprotectants dimethylsulfoxide and glycerol in final concentrations of 5% and 15% respectively. Freezing of the stromal cells on the mesh can be accomplished at initial cooling rates of -1C/min from +1C to -40C. A cooling rate of -2 to -3C/min is optimum is uti~ized until the end stage temperature of -84C is achieved. Approximately 20-25% of the stromal cells will detach from the nylon mesh.
2. inoculation with hematopoietic cells Bone marrow cells are suspended in a modified Fischer's or McCoy's 5A medium supp]emented with 5-10% fetal bovine serum and 5-10% horse serum, vitamins, hydrocortisone, glutamine and antibiotics. These cells may either be fresh -lO- 1 ~ 1 0926 or derived from a formerly cryopreserved sample which has been rapidly thawed in an 80C hot water bath. 2 to 5 X 106 cells are inoculated onto subconfluent stromal cell meshworks in 25 mm2 plastic culture flasks and grown at 33 to 34C at 5% C2 in ambient air. The relative humidity of these cultures must be greater than 90%. After 3 days the culture temperature i6 raised to 35 to 37C.
Hematopoietic cells grow in the natural pockets formed by the subconfluent stromal cells and the progenitor cells remain in the adherent layer of cells. The adherent layer are those cells attached directly to the mesh or those connected indirectly by attachment to cells that are themselves attached directly to the mesh. After 4 to 5 days, mature granulocytes, mononuclear cells, and erythrocytes appear in the non-adherent layer as observed by cytospin preparation. After 7 to 10 days, numerous hematopoietic colonies can be observed in the interstices of the mesh and are morphologically consistent with CFU-C, mixed colonies, and lymphoid colonies. Megakaryocytic growth is limited but may be observed in this matrix as well. An average culture will produce 450 to 950 CFU-C per week.
Cultures which consist of stromal cells and hematopoietic cells derived from the same individual (autologous) must be fed twice weekly. Cultures which consist of a patient's bone marrow which has been inoculated onto a stromal cell meshwork derived from another individual(s) (allogeneic) must be fed three times per week to insure adequate depopulation of mature immunocompetent cells from the non-adherent layer.
Yet another object of the present invention is to provide a means for treating diseases or conditions that destroy hçalthy bone marrow cells by aspirating bone marrow from a patient, replicating the bone marrow cells in vitro and then reinfusing the replicated marrow cells into the patient.
A still further object of the present invention is to provide a process for increasing the functional ability of a bone marrow which has been adversely affected by chemotherapy and/or radiation therapy.
Still another object of the present invention is to provide a process for the treatment of hematological malignancies and other neoplasias which metastasize to the bone marrow.
Additional objects and advantages of the present invention will be apparent to those skilled in the art by reference to the following detailed description.
The invention may be better understood by reference to the single figure of the following drawings showing a scanning electron photomicrograph at a magnification of 1. 65 thousand times showing a meshwork with bone marrow stromal cells growing thereon.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a process for treating a person whose bone marrow has been destroyed or lost its functional ability. The process comprises the steps of:
I. obtaining a bone marrow sample from a ` donor; and then II. replicating the bone marrow sample in vitro to produce a replicated bone marrow _3_ 1310q26 containing hematopoietic stem cells having marrow repopulating activity, a portion of which may be cryopreserved for later use;
and then III. infusing the replicated bone marrow containing hematopoietic stem cells into the person to restore hematopoiesis in the person.
According to the present invention, there is provided a process for treating diseases or conditions which have destroyed healthy bone marrow cells or have depressed their functional ability. The process of the present invention is especially effective in the treatment of hematological malignancies and other neoplasias which metastasize to the bone marrow.
The process of the present invention comprises the steps of: aspirating a small amount of bone marrow from a healthy patient; cryopreserving said cells; replicating the bone marrow cells in vitro to increase the number of bone marrow cells to a number sufficient for autologous reinfusion, which is greater than the number originally removed from the patient; and then reinfusing the bone marrow cells into the patient. According to one embodiment of the present invention, the bone marrow cells are cryopreserved or frozen immediately after aspiration from the patient and are subsequently thawed and replicated. According to another embodiment of the present invention, an allogeneic bone marrow transplant is performed by aspirating bone marrow from one person, replicating the bone marrow cells in vitro and then reinfusing the replicated marrow cells into another person.
The in vitro bone marrow replication system of the present invention also provides a means to monitor a patient in regard to those conditions that affect the bone marrow.
For example, a small sample of bone marrow is aspirated from _4~ U q 26 a treated cancer patient and replicated in the ln vitro system of the present invention, in order to check for a recurrence or metastasis of the original malignancy.
The in vitro bone marrow replication system is also adaptable for cytotoxicity testing of pharmaceuticals, food additives, health products, anti-neoplastic agents, and carcinogens. A proportional, scaled-down dose of the agent to be tested is added ~o the ln vitro bone marrow system of the invention and its effect on various cell types noted.
Cell types other than bone marrow cells are substituted to test a particular agent, as required.
The invention also describes a three-dimensional support for growing cells in culture. The growth of cells in the presence of this support may be further enhanced by adding proteins, glycoproteins, glycosaminoglycans, a cellular matrix, and other materials to the support itself or by coating the support with these materials. The use of a three-dimensional support allows the cells to grow in multiple layers, thus creating the three-dimensional, cell culture system of the present invention. The three dimensional support can be used to create three-dimensional, cell culture systems for bone marrow, skin, liver, and many other cell types and tissues. In one embodiment of this invention, the three-dimensional cell culture system may be transferred onto or into a living orgamism.
The three-dimensional support is preferably in the form of a mesh. Prepared supports may be preserved for later use in a three-dimensional, physiologic, cell culture system.
In an embodiment for growing hematopoietic bone marrow cells, the mesh i5 prepared by growing a layer of stromal marrow cells on the mesh until the stromal marrow cells reach subconfluency.
BRIEF DESCRIPTION OF THE DRAWING
Referring now to the single figure of the drawings, ~5~ 1 3 1 0~6 there is shown a meshwork 10 useful in the present invention.
The meshwork 10 contains warp threads 12 and woof threads 13.
As shown in the figure, stromal cells 14 can be seen growing on the threads 12, 13.
DETAILED DESCRIPTION OF THE INVENTION
Replication and Cyropreservation of Bone Marrow for Use in Bone Marrow Transplantations The present invention is directed to a process for treating diseases or conditions which destroy healthy bone marrow cells or depress their functional ability. The process is effective especially in the treatment of hematological malignancies and other neoplasias which metastasize to the bone marrow. The process of this invention is also effective in treating patients whose bone marrow has been adversely affected by chemotherapy and/or radiation therapy necessitated by a disease which does not directly affect the bone marrow. The process of the present invention further provides a means for removing and preserving bone marrow cells from a healthy patient and then replicating and reinfusing the bone marrow cells should the patient develop an illness which either destroys the bone marrow directly or whose treatment adversely affects the marrow.
In accordance with the process of the present invention, a small amount (10-15 cc bone marrow/peripheral blood suspension) is aspirated from the iliac crest of a donor. The results of the process are optimal if: 1. The individual is under 40 years of age at the time his/her marrow is cryopreserved, 2. the patient is disease-free.
This procedure may prove beneficial to certain patients with metastatic disease or hematological malignancies if "purging"
of malignant cells by physical or chemotherapeutic means can be performed prior to culturing. At present, these methods are most efficient when small numbers of cells are used, a fact which might lend itself well to the following procedure.
Methods of aspirating bone marrow from a donor are well known in the art. Examples of apparatus and processes for aspirating bone marrow from a donor can be found in U.S.
patents 4,481,946 and 4,486,188.
The bone marrow removed from the donor is then replicated or preserved for replication at a later date. If the bone marrow is to be preserved, the bone marrow can be incrementally frozen using computerized cryotechnological equipment. Fresh bone marrow/blood suspension is aliquoted in equal volumes into sterile Nunc tubes and placed in a beaker of crushed ice until the cryopreservation chamber is brought to a similar temperature (4C). Immediately prior to specimen insertion into the chamber, a solution is added to each Nunc tube using sterile technique, so that the cryoprotectants, dimethylsulfoxide and glycerol, will be in final concentrations of 7% and 5% respectively. The freezing program is initiated immediately after introduction of the specimen. Freezing program number 1 on the CryoMed Model Number 1010 controller is used.
Using this technique, the cellular viability after freezing and rapid thawing in an 80C water bath exceeds 90%
via the trypan blue exclusion method. In addition, greater than 80% of the original colony forming unit culture (CFU-C) may be recovered after freezing. Examples of systems for freezing bone marrow and biological substances in accordance with a precalculated temperature and time curve are disclosed in U.S. patents 4,107,937 and 4,117,881. Preferably, the bone marrow cells are stored in the liquid phase of liquid nitrogen at a temperature of -196C at which temperature all cellular metabolic activity has ceased.
After thawing, the bone marrow cells is reinfused into an individual or is replicated, and then reinfused.
^~ ~ * Trade Mark _7_ 1 3 1 Oq26 The process of the present invention has several advantages to a patient in need of a bone marrow transplant.
If the patient is receiving his or her own cells, this is called an autologous transplant. Such a transplant has little likelihood of rejection. Autologous transplants eliminate a ~ajor cause of bone marrow transplant rejection, that is, the graft vs. host reaction. Further, when grown in culture, hematopoietic cells may be easily separated by physical manipulation and/or enzyme treatment. This diminishes the risk of vasoocclusive disease resulting from marrow emboli. In addition, the process of the present invention allows more aggressive treatment of neoplastic disorders with chemotherapeutic agents and radiation.
Presently, the extent of these treatments is often limited by bone marrow toxicity.
In Vitro Bone Marrow Replication System The process of the present invention further comprises the step of replicating the bone marrow cells in vitro, in a system comparable to physiologic conditions.
Moreover, the bone marrow cells replicated in this system include all of the cells present in normal bone marrow, assuming all cell types were present in the original bone marrow inoculum.
The bone marrow cells, either obtained directly from the donor or retrieved from cryopreservative storage, are first separated from their reticulum by physical means.
The bone marrow cells are then grown in co-cultures with stromal components of normal marrow, which includes fibroblasts, macrophages, reticular cells, and adipocytes, on a three-dimensional support. Factors derived from media of splenic and/or hepatic (liver) macrophage cultures or from subsets of stromal cells may also be added to the culture~
Although marrow cells are capable of limited growth when cultured alone, long term growth of these cultures is ;, -8- 1 31 Oq26 possible only if stromal cells or their secretory products are present. See Long-Term sone Marrow Culture, D.G. Wright & J.S. Greenberger, eds., A.R. Liss, New York, (1984).
The present invention seeks to maximize the proliferation of multipotential hematopoietic stem cells which have the capability of repopulating bone marrow when the bone marrow has been destroyed by intrinsically or environmentally-mediated disease or by the treatment of such disease with chemotherapy and/or radiation. Stem cells which have marrow repopulating activity (MRA) have been shown to persist and replicate in the long term bone marrow cultures.
However, stem cells, hemopoietic progenitor cells, hemopoietic precusor cells all replicate and proliferate in the system of the present invention. Furthermore, differentiation can proceed in this system in a physiologic manner. For example, erythroid, myeloid, lymphoid, macrophagic, and megakaryocytic colonies, can continuously arise in the same culture system using the systems as taught by the present invention.
According to a preferred embodiment of the present invention, hematopoietic stem cell growth is accomplished as follows:
1. establishment of the stromal support layer Marrow suspensions are centrifuged at 3000 x g for 20 minutes and the white base of cells containing macrophages, fibroblasts, adipocytes, mononuclear blood cells, reticular cells, endothelial cells and other resident cells is removed. The cells are suspended in a medium called Roswell Park Memorial Institute medium number 1640 or simply "RPMI 1640~. The RPMI 1640 is purchased from GIBCO
Incorporated of Grand Island, New York, USA. The RPMI 1640 supplemented with 10% fetal bovine serum, 10% horse serum, hydrocortisone hemisuccinate, and appropriate antibiotics.
106 of these cells are plated onto sterile nylon mesh from the Tetko Corp. of New York, New York, USA in a petri dish. This mesh has been pre-cut to conform to the inside dimensions of a 25mm2 plastic culture flasks. The mesh has a sieve area of 400~m2 and a fiber diameter of lOO~m-The inoculated mesh is then wound and placed intothe culture flask containing 5 milliliters of media as previously described. The mesh unwinds inside the culture flask and completely covers the bottom of the flask. The cultures are grown at 37C in 5% C02 in ambient air at a relative humidity in excess of 90%. Stromal cells which are predominantly fibroblasts first grow along and completely encircle all of the nylon fibers before beginning to grow into the mesh openings. This process takes approximately 14 to 18 days. The degree of subconfluency of the stromal cells, should be consistent with that seen in the figure of the drawing, prior to the inoculation of hematopoietic cells.
Suspended stromal cells can be cryopreserved using the same technigue as previously described for bone marrow cells. For cryopreservation of sub-confluent cells on the mesh, the nylon mesh must be rolled and inserted into the Nunc tube containing RPMI 1640 medium with the cryoprotectants dimethylsulfoxide and glycerol in final concentrations of 5% and 15% respectively. Freezing of the stromal cells on the mesh can be accomplished at initial cooling rates of -1C/min from +1C to -40C. A cooling rate of -2 to -3C/min is optimum is uti~ized until the end stage temperature of -84C is achieved. Approximately 20-25% of the stromal cells will detach from the nylon mesh.
2. inoculation with hematopoietic cells Bone marrow cells are suspended in a modified Fischer's or McCoy's 5A medium supp]emented with 5-10% fetal bovine serum and 5-10% horse serum, vitamins, hydrocortisone, glutamine and antibiotics. These cells may either be fresh -lO- 1 ~ 1 0926 or derived from a formerly cryopreserved sample which has been rapidly thawed in an 80C hot water bath. 2 to 5 X 106 cells are inoculated onto subconfluent stromal cell meshworks in 25 mm2 plastic culture flasks and grown at 33 to 34C at 5% C2 in ambient air. The relative humidity of these cultures must be greater than 90%. After 3 days the culture temperature i6 raised to 35 to 37C.
Hematopoietic cells grow in the natural pockets formed by the subconfluent stromal cells and the progenitor cells remain in the adherent layer of cells. The adherent layer are those cells attached directly to the mesh or those connected indirectly by attachment to cells that are themselves attached directly to the mesh. After 4 to 5 days, mature granulocytes, mononuclear cells, and erythrocytes appear in the non-adherent layer as observed by cytospin preparation. After 7 to 10 days, numerous hematopoietic colonies can be observed in the interstices of the mesh and are morphologically consistent with CFU-C, mixed colonies, and lymphoid colonies. Megakaryocytic growth is limited but may be observed in this matrix as well. An average culture will produce 450 to 950 CFU-C per week.
Cultures which consist of stromal cells and hematopoietic cells derived from the same individual (autologous) must be fed twice weekly. Cultures which consist of a patient's bone marrow which has been inoculated onto a stromal cell meshwork derived from another individual(s) (allogeneic) must be fed three times per week to insure adequate depopulation of mature immunocompetent cells from the non-adherent layer.
3. variations in the in vitro bone marrow replication system Enhancing the Growth of Marrow Stromal Cells The primary rate limiting factor in the growth of marrow stromal cells is the relatively low mitotic index of 1 3 1 Oq26 the fibroblasts, included among the marrow stromal cells.
The growth of these cells and their deposition of extracellular matrix components may be enhanced by adding:
1. hydrocortisone hemisuccinate and/or 2. self-regulating growth factors derived from the medium of cultured human fetal fibroblasts which have a high rate of cell dîvision.
Attachment and growth of fibroblasts on the mesh can also be enhanced by: 1. pre-coating the mesh with solubilized type I-IV collagen, or 2. using a mesh which is coated or embedded with collagen secreted by fetal human fibroblasts or by adult fibroblasts (hereinafter referred to as "growth enhancing fibroblasts") which have been subsetted by their ability to synthesize certain collagen types. In this regard, the growth enhancing fibroblasts are lifted by mild trypsinization from the mesh upon reaching confluency (5 to 7 days for fetal human fibroblasts and 14 to 18 days for adult fibroblasts respectively) and may either be 1. inoculated with stromal marrow cells as previously described or 2. cryopreserved for future use.
In one embodiment of the invention, growth enhancing fibroblasts that are synthesizing collagen and other extracellular matrix components are grown on the mesh until they reach subconfluency. A mixture of both hematopoietic and stromal bone marrow cells are then inoculated onto the subconfluent growth enhancing fibroblast meshwork.
The methods for growing, subsetting, and cryopreserving growth enhancing fibroblasts are as follows:
a. Culture of Growth Enhancing Fibroblasts Fibroblasts are grown in ~PMI 164~ supplemented with 2-10% fetal bovine serum or 2-10% horse serum to which l~g/mL hydrocortisone hemisuccinate and 2~g/mL gentamycin, penicillin, streptomycin and fungizone have been added.
Cultures are grown at 5% C02 in ambient air at 37C with a relative humidity greater than 90%.
b. Subsettiny Growth Enhancing Fibroblasts 5.0 X 106 fibroblasts derived from the buffy coat of a bone marrow suspension, dermal fibroblasts, or fibroblasts derived from cadaver livers are plated onto microtiter wells (lmm2) and grown to confluency. These cells are lifted from the culture wells by repeated washinqs usually four to five times with Hank's balanced salt solution without Ca+t or Mg++. The matrix remaining on the microtiter plates is examined by indirect immunofluorescence utilizing monoclonal antibodies to various matrix components and fluorescein isothiocyanate-labelled, rabbit anti-mouse immunoglobulin G to ascertain the collagen types present.
The suspended cells are treated with monoclonal antibodies directed against collagen types I-IV, elastin, tropoelastin, and fibronectin to isolate sub-populations of cells capable of synthesizing each product. The cells are treated with guinea pig complement which will damage or destroy those cells to which monoclonal antibody is attached. The viable cells are re-plated onto microtiter wells as previously described, are grown to confluency, and lifted. The efficiency of the isolation technique is then verified by examining the matrix secreted by those cells with monoclonal antibodies and indirect immunofluorescence.
For optimal growth of hematopoietic cells the matrix should contain collagen types III, IV and I in an approximate ratio of 6:3:1.
c. Cryopreservation of Growth Enhancing Fibroblasts Growth enhancing fibroblasts can be cryopreserved us;ng the same techniques as previously described for stromal cells. Like the stromal cells, some of the growth enhancing fibroblasts will also detach from the mesh during freezing. This matrix, however, still contributes to the attachment of marrow stromal cells and therefore diminishes the time required for the establishment of a matrix conducive to hematopoietic cell growth.
Inoculation With Mononuclear Cells To enhance the long-term growth of bone marrow cultures, peripheral blood mononuclear cells are prepared from a heparinized suspension usin~ Ficoll-hypague or Percoll. Peripheral blood cells and bone marrow hematopoietic cells are derived from the same individual lautologous). These should be removed via venipuncture and cryopreserved at the time the bone marrow specimen is taken.
Additional peripheral blood cells could be procured from the diseased patient if needed during the culturing procedure.
However, if metastic disease is suspected, the sample must first be subjected to purging, as mentioned previously.
5 x 105 to 106 mononuclear cells (the monocyte subpopulation is the preferred cell type within the mononuclear cell layer for this step) are inoculated onto meshworks 4 to 5 days after the initial inoculation with bone marrow hematopoietic cells and every third week thereafter. This procedure enhances hematopoiesis by 10 to 13~ as observed on a weekly basis.
Perpetuation Of The Culture And Banking Of Progenitor Cells In our experience, confluent stromal cell cultures will not or at best poorly support hematopoiesis.
Indefinite growth of human hematopoietic progenitors is possible if they are provided with the necessary stromal-derived growth/regulatory factors.
For example, the initial marrow sample is dividedinto a number of aliquots containing approximately 106 hematopoietic cells. Each of these is inoculated onto a sub-confluent stromal cell meshwork. The cultures are monitored by direct observation with an inverted phase , i , , ~ . ~
* Trade Mark microscope and differential counts of the non-adherent cells as seen on the cytospin preparation after each feeding.
Prior to reaching confluency, the cultures are treated with collagenase and placed under mild ultrasonication for approximate]y 6-10 minutes. Hematopoietic cells and stromal cells are separated by density gradient methods. The hematopoietic cells are counted using a hemacytometer and approximately 50% are cryopreserved using methods described previously. The remaining 50% of the hematopoietic cells are divided into aliquots consisting of approximately 106 cells and are inoculated onto sub-confluent stromal cell cultures which have been staggered and grown in parallel.
When these begin to reach confluency, the same procedure is performed.
This technique: 1. perpetuates the growth of hematopoietic cells by providing a microenvironment which produces the required growth factors and, 2. forms a continuous bank where hematopoietic progenitors may be deposited until the numbers suitable for engraftment are achieved.
Modulation Of Hematopoietic Cell Growth With Cell Products The technology presently exists to sub-culture the various cellular components of human marrow as separate cultures. Macrophages, reticular cells, adipocytes, and fibroblasts may be grown separately and their secretory activity modified by treatment with various agents.
Modulation of fibroblasts activity has been described previously.
Hematopoiesis in long-term human marrow cultures on the three dimensional meshwork may also be modulated by secretions of extramedullary macrophages (Kupffer cells) when grown in culture in the following manner. Kupffer cells are separated from their organ stroma after pronase digestion. Briefly, tissue specimens will be incubated for t 31 ~926 1 hour in pronase solution [0.2% pronase (Calbiochem) and Geys' ~alanced Salt Solution (BSS)] while being gently agitated. The pH of the solution is maintained at 7.3 to 7.5 with lN NaOH. Deoxyribonuclease (0.5 mg) (Calbiochem) is added at 30 minute intervals during the above procedure and the resultant cell suspension is filtered and centrifuged at 350 x G for 10 minutes. The pellet is resuspended in Geys' BSS and the littoral cells (macrophages and endothelial cells) are separated from the cellular debris and mature blood cells using a Percoll (Pharmacia) gradient. The resultant cell fraction is washed 3 x 3 minutes with a modified Dulbecco's medium enriched with 10%
fetal bovine serum and plated onto plastic culture dishes at a volume containing 3 to 4 x 106 cells.
After incubation for 1 day, the non-adherent cells are removed by washing with the culture medium and the adherent cell are maintained at 33C in a gas mixture consisting of 6~ C02 in room air at over 80% relative humidity. The growth and/or secretory activity of these cells can be stimulated by: 1. varying the C02; 2 ratio, 2. treating the cultures with latex beads, 3. treating the cultures with silica, 4. adding prostaglandin E2, El or F2 to the medium, 5. supplementing the medium with interleukin 1 or interleukin 2. Macrophage secretory products may be modulated by these procedures/agents.
The medium conditioned with the secretory products-of these macrophages may be used to supplement the long-term bone marrow culture erythropoietic/granulopoietic ratio in much the same manner as occurs ln vivo.
The process of the present invention has several advantages to a patient in need of a bone marrow transplant.
Use of In Vitro Bone Marrow Replication System to Monitor a Patient's Condition In a patient with cancer or other diseases, it is * Trade ~lark t 3 t ~6 often efficacious to monitor the patient's condition by aspirating a portion of the patient's bone marrow and examining the sample. In this manner, a metastasis or recurrence may be detected before it is clinically obvious.
Patients with other conditions that are detectable by examining bone marrow cells may also be monitored in this way.
The long-term growth of cells in an aspirated bone marrow specimen using the bone marrow replication system of the present invention enhances the likelihood of the detection of clonal metas~atic cells and hematopoietic cells with chromosomal abnormalities. These cells may escape detection in a conventional smear of freshly aspirated (uncultured) bone marrow.
Use of In Vitro Bone Marrow Replication System in Cytotoxicity System The cytotoxicity to bone marrow of pharmaceuticals, anti-neoplastic agents, carcinogens, food additives, and other substances to bone marrow is tested by utilizing the in vitro bone marrow replication system of the present invention.
First, stable, growing cultures of bone marrow cells (including both stromal and hematopoietic cells) are established. Then, the cultures are exposed to varying concentrations of the test agent. After incubation with the test agents, the culture are examined by phase microscopy to determine the highest tolerated dose (HTD) - the concen-tration of test agent at which the earliest morphological abnormalities appear. Cytotoxicity testing can be performed using a variety of supravital dyes to assess cell viability in this three-dimensional system, using techniques well-known to those skilled in the art. The HTD determination provides a concentration range for further testing.
Once a testing range is established, varying concentrations of the test agent can be examined for their effect on viability, growth, and/or morphology of the different cell types constituting the bone marrow culture by means well known to those skilled in the art.
Other three-dimensional cell culture system as disclosed in the present invention may be adopted for use in cytotoxicity testing.
The Establishment of a Three-Dimensional Cell Culture System The present invention discloses a three-dimensional support and its use as the framework for a three-dimensional, multi-layer cell culture system. In previously known tissue culture systems, the cells were grown in a monolayer. Cells grown on a three-dimensional support in accordance with the present invention grow in multiple layers, forming a cellular matrix. This three-dimensional cell culture system approaches physiologic conditions found in vivo to a greater degree than previously described monolayer tissue culture systems. In one embodiment of this invention, the three-dimensional cell culture system may be transferred onto or into a living organism.
The three-dimensional support may be of any material that: 1. allows cells to attach to it (or can be modified to allow cells to attach to it) and 2. allows cells to grow in more than one layer. The three-dimensional support is a mesh in a preferred embodiment of the present invention.
It is contemplated that the three-dimension cell culture system is applicable to bone marrow, skin, liver, and many other cell types and tissues. For example, a three-dimension skin cell culture system is produced as follows:
1. Fibroblast are allowed to attach to a mesh and grow for 7-9 days, depositing co]lagen types I
1310q26 -18~
and III, as described previously in regard to the growth enhancing fibroblast used in the in vitro bone marrow replication systems;
2. Melanocytes are plated onto the treated mesh and are allowed to grow for 5 days;
3. Keratinocytes are inoculated onto subconfluent melanocytes.
Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations can be made without departing from the scope of t}le invention as described above and as claimed below.
The growth of these cells and their deposition of extracellular matrix components may be enhanced by adding:
1. hydrocortisone hemisuccinate and/or 2. self-regulating growth factors derived from the medium of cultured human fetal fibroblasts which have a high rate of cell dîvision.
Attachment and growth of fibroblasts on the mesh can also be enhanced by: 1. pre-coating the mesh with solubilized type I-IV collagen, or 2. using a mesh which is coated or embedded with collagen secreted by fetal human fibroblasts or by adult fibroblasts (hereinafter referred to as "growth enhancing fibroblasts") which have been subsetted by their ability to synthesize certain collagen types. In this regard, the growth enhancing fibroblasts are lifted by mild trypsinization from the mesh upon reaching confluency (5 to 7 days for fetal human fibroblasts and 14 to 18 days for adult fibroblasts respectively) and may either be 1. inoculated with stromal marrow cells as previously described or 2. cryopreserved for future use.
In one embodiment of the invention, growth enhancing fibroblasts that are synthesizing collagen and other extracellular matrix components are grown on the mesh until they reach subconfluency. A mixture of both hematopoietic and stromal bone marrow cells are then inoculated onto the subconfluent growth enhancing fibroblast meshwork.
The methods for growing, subsetting, and cryopreserving growth enhancing fibroblasts are as follows:
a. Culture of Growth Enhancing Fibroblasts Fibroblasts are grown in ~PMI 164~ supplemented with 2-10% fetal bovine serum or 2-10% horse serum to which l~g/mL hydrocortisone hemisuccinate and 2~g/mL gentamycin, penicillin, streptomycin and fungizone have been added.
Cultures are grown at 5% C02 in ambient air at 37C with a relative humidity greater than 90%.
b. Subsettiny Growth Enhancing Fibroblasts 5.0 X 106 fibroblasts derived from the buffy coat of a bone marrow suspension, dermal fibroblasts, or fibroblasts derived from cadaver livers are plated onto microtiter wells (lmm2) and grown to confluency. These cells are lifted from the culture wells by repeated washinqs usually four to five times with Hank's balanced salt solution without Ca+t or Mg++. The matrix remaining on the microtiter plates is examined by indirect immunofluorescence utilizing monoclonal antibodies to various matrix components and fluorescein isothiocyanate-labelled, rabbit anti-mouse immunoglobulin G to ascertain the collagen types present.
The suspended cells are treated with monoclonal antibodies directed against collagen types I-IV, elastin, tropoelastin, and fibronectin to isolate sub-populations of cells capable of synthesizing each product. The cells are treated with guinea pig complement which will damage or destroy those cells to which monoclonal antibody is attached. The viable cells are re-plated onto microtiter wells as previously described, are grown to confluency, and lifted. The efficiency of the isolation technique is then verified by examining the matrix secreted by those cells with monoclonal antibodies and indirect immunofluorescence.
For optimal growth of hematopoietic cells the matrix should contain collagen types III, IV and I in an approximate ratio of 6:3:1.
c. Cryopreservation of Growth Enhancing Fibroblasts Growth enhancing fibroblasts can be cryopreserved us;ng the same techniques as previously described for stromal cells. Like the stromal cells, some of the growth enhancing fibroblasts will also detach from the mesh during freezing. This matrix, however, still contributes to the attachment of marrow stromal cells and therefore diminishes the time required for the establishment of a matrix conducive to hematopoietic cell growth.
Inoculation With Mononuclear Cells To enhance the long-term growth of bone marrow cultures, peripheral blood mononuclear cells are prepared from a heparinized suspension usin~ Ficoll-hypague or Percoll. Peripheral blood cells and bone marrow hematopoietic cells are derived from the same individual lautologous). These should be removed via venipuncture and cryopreserved at the time the bone marrow specimen is taken.
Additional peripheral blood cells could be procured from the diseased patient if needed during the culturing procedure.
However, if metastic disease is suspected, the sample must first be subjected to purging, as mentioned previously.
5 x 105 to 106 mononuclear cells (the monocyte subpopulation is the preferred cell type within the mononuclear cell layer for this step) are inoculated onto meshworks 4 to 5 days after the initial inoculation with bone marrow hematopoietic cells and every third week thereafter. This procedure enhances hematopoiesis by 10 to 13~ as observed on a weekly basis.
Perpetuation Of The Culture And Banking Of Progenitor Cells In our experience, confluent stromal cell cultures will not or at best poorly support hematopoiesis.
Indefinite growth of human hematopoietic progenitors is possible if they are provided with the necessary stromal-derived growth/regulatory factors.
For example, the initial marrow sample is dividedinto a number of aliquots containing approximately 106 hematopoietic cells. Each of these is inoculated onto a sub-confluent stromal cell meshwork. The cultures are monitored by direct observation with an inverted phase , i , , ~ . ~
* Trade Mark microscope and differential counts of the non-adherent cells as seen on the cytospin preparation after each feeding.
Prior to reaching confluency, the cultures are treated with collagenase and placed under mild ultrasonication for approximate]y 6-10 minutes. Hematopoietic cells and stromal cells are separated by density gradient methods. The hematopoietic cells are counted using a hemacytometer and approximately 50% are cryopreserved using methods described previously. The remaining 50% of the hematopoietic cells are divided into aliquots consisting of approximately 106 cells and are inoculated onto sub-confluent stromal cell cultures which have been staggered and grown in parallel.
When these begin to reach confluency, the same procedure is performed.
This technique: 1. perpetuates the growth of hematopoietic cells by providing a microenvironment which produces the required growth factors and, 2. forms a continuous bank where hematopoietic progenitors may be deposited until the numbers suitable for engraftment are achieved.
Modulation Of Hematopoietic Cell Growth With Cell Products The technology presently exists to sub-culture the various cellular components of human marrow as separate cultures. Macrophages, reticular cells, adipocytes, and fibroblasts may be grown separately and their secretory activity modified by treatment with various agents.
Modulation of fibroblasts activity has been described previously.
Hematopoiesis in long-term human marrow cultures on the three dimensional meshwork may also be modulated by secretions of extramedullary macrophages (Kupffer cells) when grown in culture in the following manner. Kupffer cells are separated from their organ stroma after pronase digestion. Briefly, tissue specimens will be incubated for t 31 ~926 1 hour in pronase solution [0.2% pronase (Calbiochem) and Geys' ~alanced Salt Solution (BSS)] while being gently agitated. The pH of the solution is maintained at 7.3 to 7.5 with lN NaOH. Deoxyribonuclease (0.5 mg) (Calbiochem) is added at 30 minute intervals during the above procedure and the resultant cell suspension is filtered and centrifuged at 350 x G for 10 minutes. The pellet is resuspended in Geys' BSS and the littoral cells (macrophages and endothelial cells) are separated from the cellular debris and mature blood cells using a Percoll (Pharmacia) gradient. The resultant cell fraction is washed 3 x 3 minutes with a modified Dulbecco's medium enriched with 10%
fetal bovine serum and plated onto plastic culture dishes at a volume containing 3 to 4 x 106 cells.
After incubation for 1 day, the non-adherent cells are removed by washing with the culture medium and the adherent cell are maintained at 33C in a gas mixture consisting of 6~ C02 in room air at over 80% relative humidity. The growth and/or secretory activity of these cells can be stimulated by: 1. varying the C02; 2 ratio, 2. treating the cultures with latex beads, 3. treating the cultures with silica, 4. adding prostaglandin E2, El or F2 to the medium, 5. supplementing the medium with interleukin 1 or interleukin 2. Macrophage secretory products may be modulated by these procedures/agents.
The medium conditioned with the secretory products-of these macrophages may be used to supplement the long-term bone marrow culture erythropoietic/granulopoietic ratio in much the same manner as occurs ln vivo.
The process of the present invention has several advantages to a patient in need of a bone marrow transplant.
Use of In Vitro Bone Marrow Replication System to Monitor a Patient's Condition In a patient with cancer or other diseases, it is * Trade ~lark t 3 t ~6 often efficacious to monitor the patient's condition by aspirating a portion of the patient's bone marrow and examining the sample. In this manner, a metastasis or recurrence may be detected before it is clinically obvious.
Patients with other conditions that are detectable by examining bone marrow cells may also be monitored in this way.
The long-term growth of cells in an aspirated bone marrow specimen using the bone marrow replication system of the present invention enhances the likelihood of the detection of clonal metas~atic cells and hematopoietic cells with chromosomal abnormalities. These cells may escape detection in a conventional smear of freshly aspirated (uncultured) bone marrow.
Use of In Vitro Bone Marrow Replication System in Cytotoxicity System The cytotoxicity to bone marrow of pharmaceuticals, anti-neoplastic agents, carcinogens, food additives, and other substances to bone marrow is tested by utilizing the in vitro bone marrow replication system of the present invention.
First, stable, growing cultures of bone marrow cells (including both stromal and hematopoietic cells) are established. Then, the cultures are exposed to varying concentrations of the test agent. After incubation with the test agents, the culture are examined by phase microscopy to determine the highest tolerated dose (HTD) - the concen-tration of test agent at which the earliest morphological abnormalities appear. Cytotoxicity testing can be performed using a variety of supravital dyes to assess cell viability in this three-dimensional system, using techniques well-known to those skilled in the art. The HTD determination provides a concentration range for further testing.
Once a testing range is established, varying concentrations of the test agent can be examined for their effect on viability, growth, and/or morphology of the different cell types constituting the bone marrow culture by means well known to those skilled in the art.
Other three-dimensional cell culture system as disclosed in the present invention may be adopted for use in cytotoxicity testing.
The Establishment of a Three-Dimensional Cell Culture System The present invention discloses a three-dimensional support and its use as the framework for a three-dimensional, multi-layer cell culture system. In previously known tissue culture systems, the cells were grown in a monolayer. Cells grown on a three-dimensional support in accordance with the present invention grow in multiple layers, forming a cellular matrix. This three-dimensional cell culture system approaches physiologic conditions found in vivo to a greater degree than previously described monolayer tissue culture systems. In one embodiment of this invention, the three-dimensional cell culture system may be transferred onto or into a living organism.
The three-dimensional support may be of any material that: 1. allows cells to attach to it (or can be modified to allow cells to attach to it) and 2. allows cells to grow in more than one layer. The three-dimensional support is a mesh in a preferred embodiment of the present invention.
It is contemplated that the three-dimension cell culture system is applicable to bone marrow, skin, liver, and many other cell types and tissues. For example, a three-dimension skin cell culture system is produced as follows:
1. Fibroblast are allowed to attach to a mesh and grow for 7-9 days, depositing co]lagen types I
1310q26 -18~
and III, as described previously in regard to the growth enhancing fibroblast used in the in vitro bone marrow replication systems;
2. Melanocytes are plated onto the treated mesh and are allowed to grow for 5 days;
3. Keratinocytes are inoculated onto subconfluent melanocytes.
Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations can be made without departing from the scope of t}le invention as described above and as claimed below.
Claims (98)
1. A living stromal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocom-patible, non-living material formed into a three-dimen-sional structure having interstitial spaces bridged by the stromal cells.
2. The living stromal tissue of claim 1 in which the stromal cells are fibroblasts.
3. The living stromal tissue of claim 1 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.
4. The living stromal tissue of claim 1 in which the framework is composed of a biodegradable material.
5. The living stromal tissue of claim 4 in which the biodegradable material comprises cotton, poly-glycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
6. The living stromal tissue of claim 1 in which the framework is composed of a non-biodegradable material.
7. The living stromal tissue of claim 6 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.
8. The living stromal tissue of claim 4, 5, 6 or 7 in which the framework is pre-coated with collagen.
9. The living stromal tissue of claim 1, 2, 3, 4, 5, 6 or 7 in which the framework is a mesh.
10. The living stromal tissue of claim 8 in which the framework is a mesh.
11. A three-dimensional cell culture comprising parenchymal cells cultured on a living stromal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework com-posed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells.
12. The three-dimensional cell culture of claim 11 in which the stromal cells are fibroblasts.
13. The three-dimensional cell culture of claim 11 in which the stromal cells are a combination of fibro-blasts and endothelial cells, pericytes, macrophages, mono-cytes, leukocytes, plasma cells, mast cells or adipocytes.
14. The three-dimensional cell culture of claim 11 in which the framework is composed of a biodegradable material.
15. The three-dimensional cell culture of claim 14 in which the biodegradable material is cotton, polygly-colic acid, cat gut sutures, cellulose, gelatin, or dex-tran.
16. The three-dimensional cell culture of claim 11 in which the framework is composed of a non-biodegrad-able material.
17. The three-dimensional cell culture of claim 16 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacryl-ate, a polyvinyl, a polycarbonate, a polytetrafluorethyl-ene, or a nitrocellulose compound.
18. The three-dimensional cell culture of claim 14, 15, 16 or 17 in which the framework is pre-coated with collagen.
19. The three-dimensional cell culture of claim 11, 12, 13, 14, 15, 16 or 17 in which the framework is a mesh.
20. The three-dimensional cell culture of claim 18 in which the framework is a mesh.
21. A three-dimensional bone marrow culture com-prising hematopoietic cells cultured on a living stromal tissue prepared in vitro, comprising stromal cells and con-nective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells.
22. A three-dimensional skin culture comprising melanocytes and keratinocytes cultured on a living stromal tissue prepared in vitro, comprising stromal cells and con-nective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells.
23. The three-dimensional skin culture of claim 22 in which the stromal cells are confluent.
24. A three-dimensional liver culture comprising hepatocytes cultured on a living stromal tissue prepared in vitro, comprising stromal cells and connective tissue pro-teins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a bio-compatible, non-living material formed into a three-dimen-sional structure having interstitial spaces bridged by the stromal cells.
25. A method for culturing cells in vitro com-prising:
a) inoculating parenchymal cells onto a living stromal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells at-tached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimen-sional structure having interstitial spaces bridged by the stromal cells; and b) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated cells proliferate in culture.
a) inoculating parenchymal cells onto a living stromal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells at-tached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimen-sional structure having interstitial spaces bridged by the stromal cells; and b) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated cells proliferate in culture.
26. The method according to claim 25 in which the stromal cells are fibroblasts.
27. The method according to claim 25 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.
28. The method according to claim 25 in which the framework is composed of a biodegradable material.
29. The method according to claim 28 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
30. The method according to claim 25 in which the framework is composed of a non-biodegradable material.
31. The method according to claim 30 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a poly-vinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.
32. The method according to claim 28, 29, 30 or 31 in which the framework is pre-coated with collagen.
33. The method according to claim 25, 26, 27, 28, 29, 30 or 31 in which the framework is a mesh.
34. The method according to claim 33 in which the framework is a mesh.
35. A method for culturing bone marrow cells in vitro, comprising:
a) inoculating hematopoietic cells onto a liv-ing stromal tissue prepared in vitro, com-prising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially envelop-ing a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and b) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated cells proliferate in culture.
a) inoculating hematopoietic cells onto a liv-ing stromal tissue prepared in vitro, com-prising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially envelop-ing a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and b) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated cells proliferate in culture.
36. A method for culturing skin cells in vitro, comprising:
a) inoculating melanocytes and keratinocytes onto a living stromal tissue prepared in vitro, comprising stromal cells and connec-tive tissue proteins naturally secreted by the stromal cells attached to and substan-tially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and b) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated cells proliferate in culture.
a) inoculating melanocytes and keratinocytes onto a living stromal tissue prepared in vitro, comprising stromal cells and connec-tive tissue proteins naturally secreted by the stromal cells attached to and substan-tially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and b) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated cells proliferate in culture.
37. The method according to claim 36 in which the living stromal tissue comprises confluent stromal cells.
38. A method for culturing liver cells in vitro, comprising:
a) inoculating hepatocytes onto a living stro-mal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells at-tached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimen-sional structure having interstitial spaces bridged by the stromal cells; and b) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated cells proliferate in culture.
a) inoculating hepatocytes onto a living stro-mal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells at-tached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimen-sional structure having interstitial spaces bridged by the stromal cells; and b) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated cells proliferate in culture.
39. A method for testing the cytological effect of a test substance comprising:
a) exposing a three-dimensional cell culture to the test substance, in which the three-dimensional cell culture comprises parenchy-mal cells grown on a living stromal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and b) determining the effect of the test substance by measuring a change in the three-dimen-sional cell culture.
a) exposing a three-dimensional cell culture to the test substance, in which the three-dimensional cell culture comprises parenchy-mal cells grown on a living stromal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and b) determining the effect of the test substance by measuring a change in the three-dimen-sional cell culture.
40. The method of claim 39 for testing the cyto-toxicity of a test substance, wherein the effect of the test substance is determined by measuring the number of damaged or dead cells in the three-dimensional cell cul-ture, in which the number of damaged or dead cells corre-lates with the cytotoxicity of the test substance.
41. The method of claim 39 for testing the growth/regulatory activity of a test substance, wherein the effect of the test substance is determined by analyzing the cellular content of the three-dimensional cell culture, in which an increase in cell number or a cell type correlates with the growth/regulatory activity of the test substance.
42. The method of claim 39 for testing the meta-bolic activity of a test substance, wherein the effect of the test substance is determined by measuring a metabolite produced by the three-dimensional cell culture, in which an increase or decrease in the amount of metabolite produced correlates with the activity of the test substance.
43. The method of claim 39 for testing the al-lergenic effect of a test substance, wherein the three-dimensional cell cultures contain mast cells, and the ef-fect of the test substance is determined by measuring the release of a vasoactive mediator by the mast cells, in which release of the vasoactive mediator correlates with the allergenic effect of the test substance.
44. The method for testing the cytological effect of a test substance according to claim 39 in which the stromal cells are fibroblasts.
45. The method for testing the cytological effect of a test substance according to claim 39 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leu-kocytes, plasma cells, mast cells or adipocytes.
46. The method for testing the cytological ef-fect of a test substance according to claim 39 in which the framework is composed of a biodegradable material.
47. The method for testing the cytological ef-fect of a test substance according to claim 46 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin or dextran.
48. The method for testing the cytological ef-fect of a test substance according to claim 39 in which the framework is composed of non-biodegradable materials.
49. The method for testing the cytological ef-fect of a test substance according to claim 48 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocel-lulose compound.
50. The method for testing the cytological ef-fect of a test substance according to claim 46, 47, 48 or 49 in which the framework is pre-coated with collagen.
51. The method for testing the cytological ef-fect of a test substance according to claim 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 in which the framework is a mesh.
52. The method for testing the cytological ef-fect of a test substance according to claim 50 in which the framework is a mesh.
53. The method for testing the cytological ef-fect of a test substance according to claim 39 in which the parenchymal cells are hematopoietic cells.
54. The method for testing the cytological ef-fect of a test substance according to claim 39 in which the parenchymal cells are melanocytes and keratinocytes.
55. The method for testing the cytological ef-fect of a test substance according to claim 39 in which the parenchymal cells are hepatocytes.
56. A method for testing the effect of a drug, comprising:
a) exposing a three-dimensional cell culture to the drug, in which the three-dimensional cell culture comprises parenchymal cells grown on a living stromal tissue prepared in vitro, comprising stromal cells and connec-tive tissue proteins naturally secreted by the stromal cells attached to and substan-tially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and b) determining the effect of the drug on the cells in culture.
a) exposing a three-dimensional cell culture to the drug, in which the three-dimensional cell culture comprises parenchymal cells grown on a living stromal tissue prepared in vitro, comprising stromal cells and connec-tive tissue proteins naturally secreted by the stromal cells attached to and substan-tially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and b) determining the effect of the drug on the cells in culture.
57. The method for testing the effect of a drug according to claim 56 in which the stromal cells are fibro-blasts.
58. The method for testing the effect of a drug according to claim 56 in which the stromal cells are a com-bination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.
59. The method for testing the effect of a drug according to claim 56 in which the framework is composed of a biodegradable material.
60. The method for testing the effect of a drug according to claim 59 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin or dextran.
61. The method for testing the effect of a drug according to claim 56 in which the framework is composed of non-biodegradable materials.
62. The method for testing the effect of a drug according to claim 61 in which the non-biodegradable mater-ial is a polyamide, a polyester, a polystyrene, a polypro-pylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.
63. The method for testing the effect of a drug according to claim 59, 60, 61 or 62 in which the framework is pre-coated with collagen.
64. The method according to claim 56, 57, 58, 59, 60, 61 or 62 in which the framework is a mesh.
65. The method for testing the effect of a drug according to claim 59, 60, 61 or 62 in which the framework is a mesh and is pre-coated with collagen.
66. The method for testing the effect of a drug according to claim 56 in which the parenchymal cells are hematopoietic cells.
67. The method for testing the effect of a drug according to claim 56 in which the parenchymal cells are melanocytes and keratinocytes.
68. The method for testing the effect of a drug according to claim 56 in which the parenchymal cells are hepatocytes.
69. A cytological testing apparatus, comprising a three-dimensional cell culture positioned in a container to which a test substance can be added, in which the three-dimensional cell culture comprises parenchymal cells cul-tured on a living stromal tissue prepared in vitro, compri-sing stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substan-tially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional struc-ture having interstitial spaces bridged by the stromal cells.
70. The cytological testing apparatus of claim 69 in which the stromal cells are fibroblasts.
71. The cytological testing apparatus of claim 69 in which the stromal cells are a combination of fibro-blasts and endothelial cells, pericytes, macrophages, mono-cytes, leukocytes, plasma cells, mast cells or adipocytes.
72. The cytological testing apparatus of claim 69 in which the framework is composed of a biodegradable material.
73. The cytological testing apparatus of claim 72 in which the biodegradable material is cotton, polygly-colic acid, cat gut sutures, cellulose, gelatin or dextran
74. The cytological testing apparatus of claim 69 in which the framework is composed of a non-biodegrad-able material.
75. The cytological testing apparatus of claim 74 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacryl-ate, a polyvinyl, a polycarbonate, a polytetrafluorethyl-ene, or a nitrocellulose compound.
76. The cytological testing apparatus of claim 72, 73, 74 or 75 in which the framework is pre-coated with collagen.
77. The cytological testing apparatus of claim 69, 70, 71, 72, 73, 74 or 75 in which the framework is a mesh.
78. The cytological testing apparatus of claim 76 in which the framework is a mesh.
79. The cytological testing apparatus of claim 69, 70, 71, 72, 73, 74 or 75 in which the container is a multi-welled container.
80. The cytological testing apparatus of claim 76 in which the container is a multi-welled container.
81. The cytological testing apparatus of claim 77 in which the container is a multi-welled container.
82. The cytological testing apparatus of claim 78 in which the container is a multi-welled container.
83. The cytological testing apparatus of claim 69 in which the parenchymal cells are hematopoietic cells.
84. The cytological testing apparatus of claim 69 in which the parenchymal cells are melanocytes and keratinocytes.
85. The cytological testing apparatus of claim 69 in which the parenchymal cells are hepatocytes.
86. A method for diagnosing or monitoring a malignancy in a patient, comprising:
a) obtaining a sample of cells from the patient;
b) inoculating parenchymal cells from the sample onto a living stromal tissue prepared in vitro, comprising stromal cells and con-nective tissue proteins naturally secreted by the stromal cells attached to and sub-stantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells;
c) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated cells proliferate in culture; and d) determining the presence of malignant cells in the proliferated cells in culture.
a) obtaining a sample of cells from the patient;
b) inoculating parenchymal cells from the sample onto a living stromal tissue prepared in vitro, comprising stromal cells and con-nective tissue proteins naturally secreted by the stromal cells attached to and sub-stantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells;
c) incubating the inoculated living stromal tissue in a nutrient medium so that the inoculated cells proliferate in culture; and d) determining the presence of malignant cells in the proliferated cells in culture.
87. The method for diagnosing or monitoring a malignancy according to claim 86 in which the stromal cells are fibroblasts.
88. The method for diagnosing or monitoring a malignancy according to claim 86 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.
89. The method for diagnosing or monitoring a malignancy according to claim 86 in which the framework is composed of a biodegradable material.
90. The method for diagnosing or monitoring a malignancy according to claim 89 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin or dextran.
91. The method for diagnosing or monitoring a malignancy according to claim 86 in which the framework is composed of non-biodegradable materials.
92. The method for diagnosing or monitoring a malignancy according to claim 91 in which the non-biode-gradable material is a polyamide, a polyester, a polysty-rene, a polypropylene, a polyacrylate, a polyvinyl, a poly-carbonate, a polytetrafluorethylene, or a nitrocellulose compound.
93. The method for diagnosing or monitoring a malignancy according to claim 89, 90, 91 or 92 in which the framework is pre-coated with collagen.
94. The method according to claim 86, 87, 88, 89, 90, 91 or 92 in which the framework is a mesh.
95. The method according to claim 93 in which the framework is a mesh.
96. The method for diagnosing or monitoring a malignancy according to claim 86 in which the parenchymal cells are hematopoietic cells.
97. The method for diagnosing or monitoring a malignancy according to claim 86 in which the parenchymal cells are melanocytes and keratinocytes.
98. The method for diagnosing or monitoring a malignancy according to claim 86 in which the parenchymal cells arehepatocytes..
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US85356986A | 1986-04-18 | 1986-04-18 | |
| US3811087A | 1987-04-14 | 1987-04-14 | |
| US853,569 | 1992-03-18 | ||
| US038,110 | 1997-02-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1310926C true CA1310926C (en) | 1992-12-01 |
Family
ID=26714872
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000534951A Expired - Lifetime CA1310926C (en) | 1986-04-18 | 1987-04-16 | Process for replicating bone marrow and other tissues in vitro and using the same |
Country Status (3)
| Country | Link |
|---|---|
| CA (1) | CA1310926C (en) |
| IE (1) | IE81126B1 (en) |
| NZ (1) | NZ220038A (en) |
-
1987
- 1987-04-16 CA CA000534951A patent/CA1310926C/en not_active Expired - Lifetime
- 1987-04-16 IE IE100787A patent/IE81126B1/en not_active IP Right Cessation
- 1987-04-22 NZ NZ220038A patent/NZ220038A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NZ220038A (en) | 1990-08-28 |
| IE871007L (en) | 1987-10-18 |
| IE81126B1 (en) | 2000-03-22 |
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| MKLA | Lapsed | ||
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