IE871007L - Process for replicating bone marrow and other tissues in¹vitro and using the same - Google Patents

Process for replicating bone marrow and other tissues in¹vitro and using the same

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Publication number
IE871007L
IE871007L IE871007A IE100787A IE871007L IE 871007 L IE871007 L IE 871007L IE 871007 A IE871007 A IE 871007A IE 100787 A IE100787 A IE 100787A IE 871007 L IE871007 L IE 871007L
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Ireland
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cells
bone marrow
stromal tissue
living
culturing
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IE871007A
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IE81126B1 (en
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Brian A Naughton
Gail K Naughton
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Schmidt & Lenhardt Gmbh & Co
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Publication of IE871007L publication Critical patent/IE871007L/en
Publication of IE81126B1 publication Critical patent/IE81126B1/en

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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. [CA1310926C]

Description

811 26 -1- Stromal Tissue The present invention relates to the production of a three-dimensional living stromal tissue in vitro and its use for culturing bone marrow, skin, liver and many other cell types and tissues- The cultured cells may be 5 used for transplantation, cytotoxicity testing and in vitro testing of particular disease states.
Processes for transplanting bone marrow are known. Generally, bone marrow transplants are performed on patients who suffer from a disease, such as cancer, 10 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. 15 Known 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 20 major cause of bone marrow transplant failure is vasoocclusive disease resulting from the formation of marrow emboli, procedures for the removal and storage of a persons7 marrow prior to combined chemotherapy and radiation and reinfusion of that marrow currently exist 25 (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 first removed.
One aspect of the invention provides a three-30 dimensional living stromal tissue prepared in vitro. comprising stromal cells and connective tissue proteins secreted by the stromal cells, grown on a biocompatible non-living three-dimensional support, wherein the 8112-6 -2- stromal cells are attached to and encircle the support to form a three-dimensional structure.
Further aspects provide a method of cell culture comprising bone marrow, skin or liver cells cultured on 5 the living stromal tissue, and corresponding uses of said cultured cells.
The invention describes a three-dimensional 'living stromal tissue for growing cells in culture. The growth of cells in the presence of this living stromal tissue 10 may be further enhanced by adding proteins, glycoproteins, glycosaminoglycans, a cellular matrix, and other materials to the stromal tissue itself or by coating a non-living three-dimensional support upon which the stromal tissue grows, with these materials. 15 The use of a three-dimensional living stromal tissue allows the cells to grow in multiple layers, thus creating the three-dimensional, cell culture system of the present invention. The three-dimensional living stromal tissue can be used to create three-dimensional, 2 0 cell cultures 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 organism. 25 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 is prepared by 30 growing a layer of stromal marrow cells on the mesh until the stromal marrow cells reach subconfluency.
The invention will now be described by way of example only with reference to its applications to bone marrow, it being understood however, that the invention 35 is applicable to many other cell types and tissues.
A method of culturing bone marrow for use in treating a person whose bone marrow has been destroyed -3- or lost its functional ability comprises the steps of: I. obtaining a bone marrow sample from a donor; and then II. culturing the bone marrow sample in vitro to 5 produce a cultured bone marrow containing hematopoietic stem cells having marrow repopulating activity, a portion of which may be cryopreserved for later use; and then 10 III. transplanting the cultured bone marrow containing hematopoietic stem cells into the person to restore hematopoiesis in the person.
Also, there is provided a use for treating diseases or conditions which have destroyed healthy bone marrow 15 cells or have depressed their functional ability. This is especially effective in the treatment of hematological malignancies and other neoplasias which metastasize to the bone marrow.
One embodiment of the present invention comprises 20 the steps of: aspirating a small amount of bone marrow from a healthy patient; cryopreserving said cells; culturing the bone marrow cells in vitro to increase the number of bone marrow cells to a number sufficient for autologous transplantation, for instance reinfusion, 25 which is greater than the number originally removed from the patient; and then reinfusing the bone marrow cells into the patient. According to another embodiment of the present invention, the bone marrow cells are cryopreserved or frozen immediately after aspiration 3 0 from the patient and are subsequently thawed and replicated. According to another embodiment of the present invention, an allogenic bone marrow transplant is performed by aspirating bone marrow from one person, -4- replicating the bone marrow cells in vitro and then reinfusing the replicated bone marrow cells into another person.
The in vitro bone marrow replication system also 5 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 a treated cancer patient and replicated in the in vitro system of the present .invention, in order to check for a 10 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 15 the agent to be tested is added to the in 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. 20 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. 25 Specific embodiments of the present invention will now be described by way of example only.
Referring now to the single figure of the drawings, there is shown a meshwork 10 useful in the present invention. The meshwork 10 contains warp threads 12 and 30 woof threads 13. As shown in the figure, stromal cells 14 can be seen growing on the threads 12, 13.
Replication and Cvropreservation of Bone Marrow for Use in Bone Marrow Transplantations In one embodiment the present invention is directed -5- to the culture of bone marrow cells for the manufacture of a transplant for treating diseases or conditions which destroy healthy bone marrow cells or depress their functional ability. This is effective especially in the 5 treatment of hematological malignancies and other neoplasias which metastasize to the bone marrow.' 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 10 does not directly affect the bone marrow. 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 15 destroys the bone marrow directly or whose treatment adversely affects the marrow.
In accordance with the present invention, a small amount (10-15 cc bone marrow/peripheral blood suspension) is aspirated from the iliac crest of a 20 donor. The results of the process are optimal if: 1. The individual is under 4 0 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 25 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 30 procedures. 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 35 cultured, i.e. replicated, or preserved for replication -6~ 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 5 into sterile Nunc tubes and placed in a beaker of crushed ice until the cryopreservation chamber is brought to a similar temperature (4*C). Immediately prior to specimen insertion into the chamber# a solution is added to each Nunc tube using sterile technique, so 10 that the cryoprotectants, dimethylsulfoxide and glycerol, will be 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 15 1010 controller is used.
Using this technique, the cellular viability after freezing and rapid thawing in an 80 CC water bath exceeds 90% via the trypan blue exclusion method. In addition greater than 80% of the original colony forming unit 20 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 25 cells are stored in the liquid phase of liquid nitrogen at a temperature of -196*C at which temperature all cellular metabolic activity has ceased.
After thawing, the bone marrow cells are reinfused into an individual or are replicated, and then 30 reinfused.
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 35 little likelihood of rejection. Autologous transplants eliminate a major cause of bone marrow transplant -7— 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 5 vasoocclusive disease resulting from marrow emboli. In addition, 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. 10 In Vitro Bone Marrow Replication System The method of the present invention 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 15 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 20 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. 25 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 30 is -8- possible only if stromal cells or their secretory products are present. See Long-Term Bone Marrow Culture, O.G. Wright l J.S. Greenberger, eds*, A.R. Liss, New York, (1984).
The present invention seeks to maximize the 5 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-*ediated disease or by the treatment of such disease with chemotherapy and/or radiation. Stem cells which 10 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, 15 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. 20 According to a preferred embodiment of the present invention, hematopoietic stem cell growth is accomplished as follows: 1. establishment of the stromal support layer 25 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 30 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. 35 10® of these cells are plated onto sterile nylon mesh from the Tetko Corp. of New York, New York# 0SA in a petri dish. This mesh has been pre-cut to conform to the inside dimensions of a 25mm plastic culture flasks. The mesh has a sieve area of 400um* and a fiber diameter of lOOum.
The inoculated mesh is then wound and placed into the 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 37*C in S% CO^ in ambient air at a relative humidity in excess of 90%. Stromal cells which are predominantly fibroblasts first grow along and 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 technique 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 -1*C/nin from +1*C to -40*C. A cooling rate of -2 to -3*C/min is optimum is utilized until the end stage temperature of -84 *C is achieved. Approximately 20-25% of the stromal cells will detach from the nylon mesh. 2. inoculation with hematopoietic cells Bone marrow ceils are suspended in a modified Fischer's or McCoy's 5A medium suppJe»ented with 5-10% fetal bovine serum and S-10% horse serum, vitamins, hydrocortisone, glutamine and antibiotics. These cells may either be fresh* or derived froo a formerly cryopreserved sample which has been rapidly thawed in an 80°C hot water bath. 2 to 5 X 106 cells are inoculated onto subconfluent stromal cell meshworks 2 in 25 nan plastic culture flasks and grown at 33 to 34*C at 5% C(>2 in ambient air. The relative humidity of these cultures must be greater than 90%. After 3 days, the culture temperature is raised to 35 to 37*C.
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 front 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 -lithe 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 division.
Attachment and growth of fibroblasts on the mesh can also be enhanced bys 1. pre-coating the mesh with solubilized type I-IV collagen, or 2. using a mesh which is 10 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 ^5 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. 20 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 25 inoculated onto the subconfluent growth enhancing fibroblast meshwork.
The methods for growing, subsetting, and cryopreserving growth enhancing fibroblasts are as follows: 30 a. Culture of Growth Enhancing Fibroblasts Fibroblasts are grown in RPMI 1640 supplemented J with 2-10% fetal bovine serum or 2-10% horse serum to which lug/mL hydrocortisone hemisuccinate and 2yg/mL gentamycin, penicillin, streptomycin and fungizone have been added. 35 Cultures are grown at 5% C0j in ambient air at 37°C with a -12- relative humidity greater than 90%. b. Subsetting Growth Enhancing Fibroblasts 5.0 X 106 fibroblasts derived from the buffy coat 5 of a bone marrow suspension, dermal fibroblasts/ or fibroblasts derived from cadaver livers are plated -onto 2 microtiter wells (1mm ) and grown to confluency. These cells are lifted from the culture wells by repeated washings usually four to five tiroes with Hank's balanced salt solution 10 without Ca"4"* 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. 15 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 20 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 25 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. 30 c. Cryopreservation of Growth Enhancing Fibroblasts Growth enhancing fibroblasts can be cryopreserved using 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 P5 freezing. This matrix, however, still contributes to the -13- attachment of marrow stromal cells and therefore diminishes the time required for the establishment of a matrix conductive to hematopoietic cell growth.
Inoculation With Mononuclear Cells 5 To enhance the long-term growth of bone marrow cultures, peripheral blood mononuclear cells are . prepared from a heparinized suspension using Ficoll-hypague or Percoll. Peripheral blood cells and bone marrow hematopoietic cells are derived from the same 10 individual (autologous) . 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 15 metastatic 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 20 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 25 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 30 stromal-derived growth/regulatory factors.
For example, the initial marrow sample is divided into a number of aliquots containing approximately 106 hematopoietic cells. Each of these is inoculated onto a sub-confluent stromal cell meshwork. The cultures are 35 monitored by direct observation with an inverted phase 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 approximately 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 10® 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 prev iously.
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 1 hour in pronase solution [0.2% pronase (Calbiochem) and Geys' Balanced Salt Solution (BSS)] while being gently agitated. The pH of the solution is maintained at 7.3 to 7.5 with IN 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 nature blood cells using a Percoll (Pharmacia) gradient. The resultant cell fraction is washed 3x3 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 10fi 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 33 *C in a gas mixture consisting of 6% CO^ in room air at over 80% relative humidity. The growth and/or secretory activity of these cells can be stimulated by: 1. varying the CO^.- 0^ ratio, 2. treating the cultures with latex beads, 3. treating the cultures with silica, 4. adding prostaglandin or F2o 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 _iri 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 t In a patient with metastatic (e.g. Cancer) or other diseases, it is -16- often efficacious to monitor the patient's condition by aspirating a portion of the patient's bone narrow and examining the sample. In this manner, a metastasis or recurrence ma'y 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 10 the present invention enhances the likelihood of the detection of clonal metastatic cells and hematopoietic cells with chromosomal abnormalities. These cells may escape detection in a conventional smear of freshly aspirated (uncultured) bone marrow. 15 20 25 30 35 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 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 concentration 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 -17- 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 systems 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 living stromal tissue 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. U.S. 3,997,396 discloses a method of propagating and maintaining cells of the external surface of a hollow fibre membrane. Cells grown on a three-dimensional living stromal tissue 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 living stromal may be grown on any three-dimensional biocompatible non-living support 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-dimensional cell culture system is applicable to bone marrow, skin, or liver, and any other cell types and tissues. For example, a three-dimension skin cell culture system is produced as follows: 1. Fibroblast are allowed to attached to a mesh and grown for 7-9 days, depositing collagen types I and III# as described previously in regard to the growth enhancing fibroblast used in the in vitro bone marrow replication systems; Melanocytes are plated onto the treated xnesh and are allowed to grow for 5 days; Keratinocytes are inoculated onto.subconfluent melanocytes.

Claims (17)

1. CLAIMS -19- 1. A three-dimensional living stromal tissue prepared in vitro. comprising stromal cells and connective tissue proteins secreted by the stromal cells, grown on a biocompatible non-living three-dimensional support, wherein the stromal cells are attached to and encircle the support to form a three-dimensional structure.
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, macrophages, reticular cells, mononuclear blood cells or adipocytes.
4. The living stromal tissue of any preceding claim in which the support is pre-coated with collagen.
5. The living stromal tissue of any preceding claim in which the support is a mesh.
6. The living stromal tissue of claim 5 wherein the mesh is formed of nylon.
7. A three-dimensional cell culture comprising cells cultured on a living stromal tissue according to any preceding claim, the cells being bone marrow, skin or liver cells.
8. A three-dimensional bone marrow culture comprising hematopoietic cells cultured on a living stromal tissue according to any of claims l to 6. -20-
9. A three-dimensional skin culture comprising melanocytes and keratinocytes cultured on a living stromal tissue according to any of claims 1 to 6. 5
10. The three-dimensional skin culture of claim 9 in which the stromal cells are confluent.
11. A three-dimensional liver culture comprising hepatocytes cultured on a living stromal tissue 10 according to any of claims 1 to 6.
12. A method for culturing cells in vitro comprising: (a) inoculating bone marrow, skin or liver cells onto a living stromal tissue according to any of 15 claims 1 to 6; and (b) culturing the inoculated living stromal tissue so that the inoculated cells grow. 20 25
13. A method for culturing bone marrow cells in vitro comprising: (a) inoculating hematopoietic cells onto a living stromal tissue according to any of claims 1 to 6; and (b) culturing the inoculated living stromal tissue so that the inoculated cells grow.
14. A method for culturing skin cells in vitro, comprising: (a) inoculating melanocytes and keratinocytes onto a living stromal tissue according to any of claims 30 1 to 6; and (b) culturing the inoculated living stromal tissue so that the inoculated cells grow. 35 - 21 - 10 30
15. The method according to claim 14 in which the living stromal tissue comprises confluent stromal cells.
16. A method for culturing liver cells in vitro comprising: (a) inoculating hepatocytes onto a living stromal tissue according to any of claims 1 to 6; and (b) culturing the inoculated living stromal tissue so that the inoculated cells grow.
17. The use of bone marrow, skin or liver cells cultured in vitro for the manufacture of a transplant; in which the culturing comprises: (a) inoculating bone marrow, skin or liver cells onto 15 a living stromal tissue according to any of claims 1 to 6; and (b) culturing the inoculated living stromal tissue so that the inoculated cells grow. 20 is. The use according to claim 17 wherein the cells are for transplantation of bone marrow cells prepared by culturing hematopoietic cells in vitro so that the hematopoietic cells grow. 25 19. The use according to claim 17 of skin cells prepared by culturing melanocytes and keratinocytes in vitro so that the melanocytes and keratinocytes grow. 20. The use according to claim 17 of liver ceils prepared by culturing hepatocytes in vitro for the manufacture of a transplant. 21. The use of living stromal tissue according to any of claims 1 to 6 for the manufacture of a transplant. 35 - 22 - 10 22. A method for testing the cytotoxicity of a test substance comprising: (a) exposing a three-dimensional cell culture to the test substance, in which the three-dimensional cell culture comprises bone marrow, skin or liver cells grown on a living stromal tissue according to any of claims l to 6; and (b) determining the effect of the test substance by observing any changes in the cells. 23. The method for testing the cytological effect of a test substance according to claim 23 in which the cells are hematopoietic cells. 15 24. The method for testing the cytotoxicity of a test substance according to claim 2 3 in which the cells are melanocytes and keratinocytes. 25. The method for testing the cytotoxicity of a test 20 substance according to claim 23 in which the cells are hepatocytes. 26. A method for detecting metastatic cells comprising: (a) obtaining a sample of bone marrow, skin or liver 25 cells; (b) inoculating cells from the sample onto a living stromal tissue according to any of claims 1 to 6; (c) culturing the inoculated living stromal tissue in a nutrient medium so that the inoculated cells 30 grow; and (d) determining the presence of metastatic cells in the cells grown in culture. 27. 35 The method for detecting metastatic cells according to claim 27 in which the cells are hematopoietic cells. - 23 - 28. The method for detecting metastatic cells according to claim 27 in which the cells are melanocytes and keratinocytes. 5 29. The method for diagnosing a chromosomal abnormality according to claim 27 in which the cells are hepatocytes. 30. A method of preparing in vitro a three-10 dimensional living stromal tissue comprising stromal cells and connective tissue proteins secreted by the stromal cells, which comprises growing the stromal tissue on a biocompatible non-living three-dimensional support, whereby the strcnal cells are attached to and 15 encircle the support to form a three-dimensional structure. 31. The method of claim 30 in which the stromal cells are fibroblasts. 20 32. The method of claim 20 in which the stromal cells are a combination of fibroblasts and endothelial cells, macrophages, reticular cells, mononuclear blood cells or adipocytes. 25 33. The method of any of claims 30 to 32 in which the support is pre-coatea vizh collagen. - 24 - 34. The method of any of claims 30 to 33 in which the support is a mesh. 35. The nethod of claim i4 wherein the mesh is formed 5 of nylon. 36. A three-dimensional living stromal tissue as claimed in claim 1 substantially as described herein with reference to the Examples and/or the accompanying drawings. 10 37. A method for culturing cells as claimed in any of claims 12 to 16 substantially as described herein with reference to the Examples and/or the accompanying drawings. 38. The use of cells as claimed in any of claims 17 to 21 substantially as described herein with reference to the Examples and/or the accompanying drawings. 15 39. A method as claimed in any of claims 22 to 29 substantially as described herein with reference to the Examples and/or the accompanying drawings. 40. A method of preparing tissue as claimed in claim 30 substantially as described 20 herein with reference to the Examples and/or the accompanying drawings. 41. A living stromal tissue whenever prepared by a method as claimed in any of claims 30 to 35 or 40. 25 TOMKINS & CO.
IE100787A 1986-04-18 1987-04-16 Stromal tissue IE81126B1 (en)

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US85356986A 1986-04-18 1986-04-18
US3811087A 1987-04-14 1987-04-14

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IE871007L true IE871007L (en) 1987-10-18
IE81126B1 IE81126B1 (en) 2000-03-22

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CA1310926C (en) 1992-12-01
NZ220038A (en) 1990-08-28

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