CN101558151B - The purposes that methods for cell expansion and the cell produced by this and conditioned medium are used for the treatment of - Google Patents

The purposes that methods for cell expansion and the cell produced by this and conditioned medium are used for the treatment of Download PDF

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CN101558151B
CN101558151B CN200780018851.7A CN200780018851A CN101558151B CN 101558151 B CN101558151 B CN 101558151B CN 200780018851 A CN200780018851 A CN 200780018851A CN 101558151 B CN101558151 B CN 101558151B
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cell
placenta
asc
cell mass
adherent
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CN101558151A (en
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S·梅雷茨基
Z·阿伯曼
O·伯格
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Prairie Biotechnology Co ltd
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Pluristem Ltd
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Abstract

The invention provides methods for cell expansion.Described method cultivates the adherent cell from placenta and fatty tissue under being included in the three-dimensional cultivation condition of sustenticular cell amplification.

Description

The purposes that methods for cell expansion and the cell produced by this and conditioned medium are used for the treatment of
invention field and background of invention
The present invention relates to methods for cell expansion, the cell mass that produces by this and uses thereof.The present invention relates to amplification particularly from the method for the adherent cell (adherent cell) of placenta or fatty tissue (through in whole PCT) and such as the therepic use of hematopoietic stem cell transplantation.
In growing medical circle, in order to cell immigration and organizational project object more and more need a large amount of adult stem.In addition, adult stem therapy is developed just continuously and is used for the treatment of and cures various illness, such as hematopoietic disorder, heart trouble, Parkinson's disease, Alzheimer, apoplexy, burn, muscular dystrophy, autoimmune disease, diabetes and sacroiliitis.
Hemopoietic stem cell (HSC) is progenitor cell, and it produces all blood cell type of medullary system and lymphatic system.The immigration of the HSC transplanted and start hemoposieis and depend on HSC going back to the nest and the ability of hyperplasia in receptor's marrow.
Generally generally acknowledge that stem cell has close contact with discrete tabernacle (niche) in marrow in vivo, described tabernacle provides the molecular signal via cell contact or the common mediates stem cell differentiation differentiation of short range effect and self.These tabernacles are the parts of " hematopoiesis inducibility microenvironment " (HIM), are made up of medullary cell and scavenger cell, inoblast, adipocyte and endotheliocyte.Medullary cell keeps HIM functional completeness by extracellular matrix (ECM) albumen and basement membrane composition providing promotion cell contact.They are also provided for controlling hematopoetic cell differentiation and the various solubility needed for propagation or the resident sexual cell factor.
Need the interaction between HSC and matrix to protect HSC viability and to prevent it from breaking up.After HSC transplants, the HSC of transplanting must go back to the nest marrow (BM) microenvironment residing in suitable tabernacle before its propagation and differentiation.In the process of going back to the nest, the HSC of transplanting leaves blood flow, and by arriving special tabernacle to shift through BM endothelial cell barrier with chemokine gradient.Then donor HSC must go back to the nest hematopoiesis tabernacle, and they run into the microenvironment more favourable to its division there, there HSC and mesenchymal cell, unified physics and chemistry can be set up continuously between ECM and the somatomedin of secretion contact.All these processes relate to the molecule of series of complex, such as cytokine, chemokine, hormone, steroid, extracellular matrix protein, somatomedin, cell-cell interaction albumen, adhesion protein and stromatin.
The total cellular score being moved into the special tabernacle of BM forms HSC and transplants successfully basis.In order to complete immigration, be transplanted to sanguimotor donor HSC should go back to the nest produce functional hemopoietic colony (hematopoiesis foci) to it receptor's marrow in.The number of these stoves can be multiplied by its immigration the long-pending of efficiency from the HSC sum of infusion and calculate.
It is that these cells survival rate in recipient's system is low that HSC transplants one of subject matter related to.Sufficient proof, the HSC that intravenously is transplanted is removed and is manifested in BM in several minutes after it is inculcated from circulation.3-5 hour after HSC transplants, donor's cells [Askenasy etc. 2002, the hematopoietic cell of transplanting grow thickly in vivo (Transplanted hematopoietic cells seed in clusters in recipientbone marrow in vivo) .Stem Cells.20:301-10 in receptor's marrow] is can't detect in the peripheral blood of receptor.Transplanted cells a large amount of soon after inculcating is destroyed.Therefore, it is low that receptor's marrow builds group validity, after transplanting, institute's infused cells of detecting in receptor BM for 2-3 days is only 1-5% [Kerre etc. 2001, CD34+38+ and CD34+38-two kinds of cell-specifics is gone back to the nest NOD/LtSZ scid/scid mouse bone marrow cells but show different amplification kinetics (Both CD34+38+ and CD34+38-cells homespecifically to the bone marrow of NOD/LtSZ scid/scid mice but showdifferent kinetics in expansion) .J Immunol.167:3692-8; Jetmore etc. 2002, be transplanted to the efficiency of going back to the nest of people CD34 (+) cell of the static and circulation in conditioning NOD/SCID receptor, cell cycle kinetics and survival rate (Homing efficiency, cell cyclekinetics, and survival of quiescent and cycling human CD34 (+) cellstransplanted into conditioned NOD/SCID recipients) .Blood.99:1585-93].
Mesenchyma stromal cells (Mesenchymal Stromal Cell) (MSC) is foreign cell group, can be divided into dissimilar ripe mesenchymal cell.These cells are subject to the impact of various biologically active factors and are divided into reticuloendothelial cell, inoblast, adipocyte and osteoprogenitor cells (osteogenic precursor cell).
MSC supports that the purposes that HSC moves into is known in the art.Some publications are verified when with mescenchymal stem cell co-transplantation, immigration efficiency higher [Gurevitch etc. 1999, allogeneic or xenogeneic bone marrow transplantation (the Transplantationof allogeneic or xenogeneic bone marrow within the donor stromalmicroenvironment) .Transplantation.68:1362-8 in donor matrices microenvironment of HSC; Fan etc. 2001, by trans-portal vein injection of bone marrow cell successful implementation allogeneic bone marrow transplantation (BMT): stroma cell is as cell (Successful allogeneic bone marrow transplantation (BMT) by injection of bone marrow cells via portal vein:stromal cellsasBMT-facilitating cells) the .Stem Cells.19:144-50 promoting BMT].Also demonstrate and move into co-transplantation human mesenchymal stem cell in model people sheep and cause promoting that people HSC is fitted together to the long-term immigration of BM in animal [Almeida-Porada etc. 2000, occur people donor's cells in the circulating cycle in early days by causing in the tire sheep before people's stroma cell ancestors co-transplantation to immunity and time point after a while after the transfer improves cell levels (the Co-transplantation of human stromal cellprogenitors into preimmune fetal sheep results in early appearance ofhuman donor cells in the circulation and boosts cell levels in bone marrowat later time points after transplantation) .Blood.95:3620-7 in marrow].Find to inject HSC and mescenchymal stem cell acceleration hemopoietic [Zhang etc. 2004, Stem Cells.22:1256-62] simultaneously.Recently, these find the animal model-macaque expanding to nearlyer edge.When HSC and the mescenchymal stem cell of co-transplantation monoploid identity (haploidentical), prove and facilitate HSC immigration [Liu etc. 2005, Zhonghua Xue Ye Xue Za Zhi.26:385-8].There was reported recently and use mescenchymal stem cell to promote that HSC moves into human subjects [KocON, J Clin Oncol.2000; 18:307-316:Lazarus HM, Biol Blood MarrowTransplant.2005 May; 11 (5): 389-98].
Obviously, MSC is the contribution that hematopoiesis moves into: produce and help to mediate and balance the supportive cytokine of HSC of the going back to the nest of transplanted HSC, self and directed potential; Restore impaired HSC go back to the nest and breed required hematopoieticmicroenviron-ment; And suppress the T cell (graft versus host disease (GVH disease) (GvHD) can be caused) being derived from donor, [Charbord P. and Moore, K., Ann.N.Y.Acad.Sci.1044:159-167 (2005); United States Patent (USP) the 6th, 010, No. 696; No. 6555374].Such as, [Maitra B etc. in the research of Maitra, Bone Marrow Transplant.33 (6): 597-604. (2004)] find in NOD-SCID mouse model, human mesenchymal stem cell supports unrelated donor hemopoietic stem cell and downtrod t cell activation, shows that the MSC of the non-blood people's of being derived from marrow can improve allotransplantation result.
Use a major obstacle of MSC to be the cell mass being difficult to be separated these a large amount of normal presences, it is difficulty technically and expends large, and part is because cell quantity is limited.It is marrow that MSC the most significantly originates, but relates to the shortcoming that the remarkable inconvenience obtaining Bone marrow aspirates and biopsy risk becomes these methods.Human embryo and fetus form independent life this are generally held viewpoint and makes Human embryo have query as source of human stem cell, turn increase the problem in religion and ethics on the logistical difficulties existed.
Recently the stem cell of attempting finding to gather in the crops from alternative source.Such alternative source has such as: fatty tissue, hair follicle, testis, people's olfactory mucosa, embryonic yolk sac, placenta, adolescent skin and blood (such as Cord blood even menstrual blood).But, be used for the treatment of still restricted and normally bothersome with research purpose from the stem cell of alternative source results q.s, it relates to such as from donor subject or patient's harvested cell or tissue, vitro culture and/or propagated cell, dissection (dissection) etc.
Placenta is considered to one of most possible source of stem cell, does not relate to any inconvenience or ethics constraint.Find that the MSC being derived from placenta has the characteristic similar to the MSC being derived from BM.Their energy plastic adherent, express CD105, CD73 and CD90 membrane marker, and lack the expression of CD45, CD34, CD14, CD19 and HLA-DR surface molecular.But do not resemble the MSC being derived from BM, the extremely low limit of the MSC (placentaderived-MSC, PD-MSC) being derived from placenta through interferon-γ process adjusts HLA-DR on the ground.In addition, PD-MSC cell demonstrates the immunosuppressive properties of raising under interferon-γ exists.(Chang CJ, Yen ML, Chen YC, Chien CC, Huang HI, Bai CH, Yen BL. is derived from immunosuppressive properties (Placenta-derivedMultipotent Cells exhibit immunosuppressive properties that are enhancedin the presence of interferon-gamma) the .Stem Cells.2006 Nov that placenta multipotential cell demonstrates raising under interferon-γ exists; 24 (11): 2466-77.).
Except MSC mark, PD-MSC also demonstrates unique ESC surface markers SSEA-4, TRA-1-61 and TRA-1-80, points out these may be very original cell.(Yen BL, Huang HI, Chien CC, Jui HY, Ko BS, Yao M, Shun CT, YenML, Lee MC, Chen YC. is separated multipotential cell (Isolation ofmultipotent cells from human term placenta) .Stem Cells.2005 from the mature placenta of people; 23 (1): 3-9).In addition, PD-MSC (fetal origin) but not the MSC being derived from BM is positive (Chang CJ for human leucocyte antigen (HLA)-G (HLA) in cell, Yen ML, Chen YC, Chien CC, Huang HI, Bai CH, Yen BL, immunosuppressive properties (Placenta-derived Multipotent Cells exhibitimmunosuppressive properties that are enhanced in the presence ofinterferon-gamma) the .Stem Cells.2006 Nov that placenta multipotential cell demonstrates raising is derived under interferon-γ exists, 24 (11): 2466-77.).
Research shows that the amplification potential of PD-MSC is significantly higher than MSC (the Yen BL being derived from adult BM, Huang HI, Chien CC, Jui HY, Ko BS, Yao M, Shun CT, YenML, Lee MC, Chen YC, is separated multipotential cell (Isolation ofMultipotent cells from Human Term Placenta) .Stem Cells.2005 from the mature placenta of people; 23 (1): 3-9; M.J.S.de Groot-Swings, Frans H.J.Claas, Willem E.Fibbe and Humphrey H.H.Pieternella S.in ' t Anker, Sicco A.Scherjon, Carin Kleijburg-van der Keur, Godelieve. mescenchymal stem cell (Placenta Isolation of Mesenchymal Stem Cells ofFetal or Maternal Origin from Human) the .Stem cells of placenta isolating fetal or maternal source from people, 2004; 22; 1338-1345).In addition, the adherent cell being derived from placenta can be divided into scleroblast, adipocyte and chondroblast.Find that the MSC being derived from placenta prevents Cord blood (UCB) lymphopoiesis as the MSC being derived from BM, this shows combined transplantation HSC and is derived from placenta (PD)-MSC to reduce the potential graft versus host disease (GVH disease) of receptor (GvHD) [Li CD etc., Cell Res.Jul; , and hematopoiesis support [Zhang Yi etc., Chinese Medical Journal117 (6): 882-887 (2004)] can be strengthened 15 (7): 539-47 (2005)].Placenta is instructed in such as WO00/73421 as the application that amniotic epithelial cells is originated, but it is still pretty troublesome to obtain these cells, and MSC productive rate is extremely low.
Solving MSC, to measure the other method of finiteness problem be to increase in vitro these cells [such as United States Patent (USP) the 6th, 326, No. 198 with different culture condition; No. 6030836; No. 6555374; 6th, 335, No. 195; 6th, 338, No. 942].But, the shortcoming of such method be still consuming time, need specific selection and separable programming, make these methods spend large, difficult enforcement.
Three-dimensional (3D) cell cultures more effective in output [Ma T etc., biotechnology progress (Biotechnology Progress) .Biotechnol Prog15:715-24 (1999) is found in some research; Yubing Xie, Tissue Engineering7 (5): 585-598 (2001)].The application of the 3D cultural method of simulation MSC natural surroundings is inoculated in following bio-reactor based on by these cells: the filling type bioreactor [Wendt containing Polyactive foam, D. etc., Biotechnol Bioeng 84:205-214, (2003)], radial flow filling type bioreactor [the Kitagawa etc. of tubulose poly-L-lactic acid (PLLA) porous support, Biotechnology andBioengineering 93 (5): 947-954 (2006)], with for cultivating and the plug flow reactor of amplifying candidate stem cell (United States Patent (USP) the 6th, 911, No. 201).
At United States Patent (USP) the 6th, the three-dimensional framework of adhering substrate cell is proposed, at Hosseinkhani in 022, No. 743, H etc., proposing gelatin sponge original work in [Tissue Engineering 11 (9-10): 1476-1488 (2005)] is 3D matrix.But none once mentioned that the MSC grown under these conditions was for moving into the purposes of HSC in support after HSC transplants in these researchs.Also need consuming timely to optimize various condition (such as pouring into condition) or various isolation technique for particular cell types.
The postpartum placenta that pours into is proposed in No. 7045148th, United States Patent (USP) and U.S. Patent application No. 20020123141, No. 20030032179 and No. 2005011871 as 3D reactor for cultivating the purposes of MSC.But the method is limited to be wanted until placenta is separated latter 24 hours and relate to perfusion, therefore, cell growth of trooping (mass growth) and its maintenance within the time cycle extended is impossible.
Therefore, generally generally acknowledge and need and will very advantageously possess: there is not the purposes that the methods for cell expansion of the novelty of above-mentioned restriction and the cell produced by this and conditioned medium are used for the treatment of.
invention summary
One aspect of the present invention provides methods for cell expansion, and described method cultivates the adherent cell from placenta or fatty tissue under being included in the three-dimensional cultivation condition of sustenticular cell amplification.
The present invention provides the method for Production conditions substratum on the other hand, and described method comprises: under the three-dimensional cultivation condition allowing cell amplification, cultivate the adherent cell from placenta or fatty tissue; With the conditioned medium collecting the adherent cell increased, Production conditions substratum by this.
Another aspect of the invention provides the cell mass produced according to aforesaid method.
Further aspect of the present invention provides the cell mass of separation, it comprises placenta or fatty tissue adherent cell, and wherein said adherent cells secrete is than the placenta of growth or the higher levels of at least one factor being selected from SCF, IL-6 and Flt-3 of fatty tissue adherent cells secrete in 2D cultivation.
The other aspect of the present invention provides the cell mass of separation, it comprises placenta or fatty tissue adherent cell, and the placenta of wherein said adherent cell expression ratio growth in 2D cultivates or the higher levels of of fatty tissue adherent cell expression are selected from least one protein that H2A histone family (H2AF), aldehyde dehydrogenase X (ALDH X), eukaryotic cell translation elongation factor 2 (EEEF2), net calcium binding protein 3, EF-hand shape calcium binding domains (RCN2) and calcium nurse one's health albumen 1 basic smooth muscle (CNN1).
Another aspect of the invention provides the cell mass of separation, it comprises placenta or fatty tissue adherent cell, at least one protein being selected from heterogeneity ribonucleoprotein H1 (Hnrph1) in core, CD44 isotype 2 precursor, 3 adenosine phosphate 5 phosphosulfate synthetic enzyme 2 isotype a (Papss2) and ribosome protein L 7/L a (rpL7a) of the lower expression level that the placenta of wherein said adherent cell expression ratio growth in 2D cultivates or fatty tissue adherent cell are expressed.
Further aspect of the present invention provides the cell mass of separation, and it comprises placenta or fatty tissue adherent cell, and wherein said adherent cell is characterized as higher than the immunosuppressive activity of the placenta grown in 2D cultivation or fatty tissue adherent cell.
According to the other feature of the following preferred embodiment of the invention, immunosuppressive activity comprises reduction T cell propagation.
The present invention provides pharmaceutical composition on the other hand, and it comprises the cell mass produced according to the method described above as activeconstituents.
The present invention provides pharmaceutical composition on the other hand, and it comprises the conditioned medium produced according to the method described above as activeconstituents.
Another aspect of the invention provides pharmaceutical composition, and it comprises the cell mass of the above-mentioned separation as activeconstituents.
Further aspect of the present invention provides the method can benefiting from the illness of acellular matrix graft in the object for the treatment of and having it to need, described method comprises: the adherent cell being selected from the tissue of placenta and fatty tissue giving this subject significant quantity, treats the illness can benefiting from stem cell transplantation in this object by this.
Further aspect of the present invention provides the method can benefiting from the illness of acellular matrix graft in the object for the treatment of and having it to need, described method comprises: the conditioned medium being derived from the adherent cell of the tissue being selected from placenta and fatty tissue giving this subject significant quantity, can benefit from the illness of stem cell transplantation by this in treatment target.
Further aspect of the present invention provides the method for the immunne response in the object reducing and have it to need, and described method comprises the cell mass of the separation of the claim 3,4,5,6 or 7 giving this subject significant quantity, reduces the immunne response of this object with this.
According to a feature again of described preferred embodiment, with this object of cytotherapeutic treatment.
According to a feature again of described preferred embodiment, the method comprises further and gives stem cell.
According to a feature again of described preferred embodiment, this stem cell comprises hemopoietic stem cell.
According to a feature again of described preferred embodiment, described cell and conditioned medium or adherent cell are given simultaneously.
According to a feature again of described preferred embodiment, after giving conditioned medium or adherent cell, give described cell.
According to a feature again of described preferred embodiment, this adherent cell obtains from dimensional culture.
According to a feature again of described preferred embodiment, this adherent cell is cultivated from two dimension and is obtained.
According to a feature again of described preferred embodiment, described illness is selected from: stem cell deficiencies, heart trouble, Parkinson's disease, cancer, Alzheimer, apoplexy, burn, tissue defect (loss oftissue), lose blood, anemia, autoimmune disease, diabetes, sacroiliitis, multiple sclerosis, graft versus host disease (GVH disease) (GvHD), nerve degenerative diseases, Autoimmune Encephalomyelitis (EAE), systemic lupus erythematous (SLE), rheumatoid arthritis, systemic sclerosis, Sjorgen syndrome, multiple sclerosis (MS), myasthenia gravis (MG), Ge-Ba syndrome (GBS), Hashimoto thyroiditis (HT), Graves disease, insulin-dependent diabetes (IDDM) and inflammatory bowel.
According to a feature again of described preferred embodiment, described dimensional culture comprises 3D bio-reactor.
According to a feature again of described preferred embodiment, described bio-reactor is selected from plug flow reactor, continuous agitator tank bio-reactor and fixed-bed bioreactor.
According to a feature again of described preferred embodiment, cell cultures is carried out in continuous flow substratum.
According to a feature again of described preferred embodiment, dimensional culture comprises adhesion material and is selected from: polyester, polyalkenes, poly-fluorine vinylchlorid, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibre, ceramic particle, matrigel (matrigel), extracellular matrix components, collagen, poly-L-lactic acid and inert metal fiber.
According to a feature again of described preferred embodiment, described cultivation at least carries out 3 days.
According to a feature again of described preferred embodiment, described cultivation at least carries out 3 days.
According to a feature again of described preferred embodiment, converge (confluence) just complete described cultivation until adherent cell reaches at least 60%.
According to a feature again of described preferred embodiment, described illness can benefit from the promotion moved into hemopoietic stem cell.
According to a feature again of described preferred embodiment, described adherent cell comprises the positive marker expression array (marker expressionarray) being selected from CD73, CD90, CD29 and CD105.
According to a feature again of described preferred embodiment, described adherent cell comprises the negative marker expression array being selected from CD45, CD80, HLA-DR, CD11b, CD14, CD19, CD34 and CD79.
According to a feature again of described preferred embodiment, described adherent cells secrete is than the higher levels of at least one factor being selected from SCF, Flt-3 and IL-6 of the adherent cells secrete from placenta or fatty tissue of growth in cultivating at 2D.
According to a feature again of described preferred embodiment, described adherent cell expression ratio the higher levels of of adherent cells secrete from placenta or fatty tissue of growth in 2D cultivates is selected from least one protein that H2A histone family (H2AF), aldehyde dehydrogenase X (ALDH X), eukaryotic cell translation elongation factor 2 (EEEF2), net calcium binding protein 3, EF-hand shape calcium binding domains (RCN2) and calcium nurse one's health albumen 1 basic smooth muscle (CNN1).
According to a feature again of described preferred embodiment, at least one protein being selected from heterogeneity ribonucleoprotein H1 (Hnrph1) in core, CD44 isotype 2 precursor, 3 adenosine phosphate 5 phosphosulfate synthetic enzyme 2 isotype a (Papss2) and ribosome protein L 7/L a (rpL7a) of described adherent cell expression ratio lower expression level of the adherent cells secrete from placenta or fatty tissue of growth in 2D cultivates.
According to a feature again of described preferred embodiment, described adherent cell or substratum be characterized as than 2D cultivate in growth placenta or fatty tissue adherent cell immunosuppressive activity higher.
According to a feature again of described preferred embodiment, described immunosuppressive activity comprises reduction T cell propagation.
According to a feature again of described preferred embodiment, described cell comprises the cell with stromal stem cell phenotype
According to a feature again of described preferred embodiment, described stromal stem cell phenotype comprises T cell inhibit activities.
According to a feature again of described preferred embodiment, described stromal stem cell phenotype comprises hemopoietic stem cell and supports active.
According to a feature again of described preferred embodiment, the purposes of above-mentioned cell mass is the medicine transplanted for the preparation of qualification.
The purposes that the present invention is used for the treatment of by providing novel methods for cell expansion and the cell produced by this and conditioned medium, successfully solves the shortcoming in current known structure (configuration).
Unless otherwise defined, otherwise all scientific and technical terminologies used herein have and usually understand identical implication with skilled person belonging to the present invention.In the present invention's practice or test, although can use and similar or suitable method described herein and material, suitable method and material are as mentioned below.In case of conflict, comprise patent specification defined herein will play a leading role.In addition, material, method and embodiment are only illustrative, are not intended to limit.
accompanying drawing is sketched
The present invention sets forth in this by means of only the example relevant with accompanying drawing.It is emphasised that, to be shown by embodiment in this detailed content relevant with concrete accompanying drawing and be only intended to illustrative the preferred embodiments of the invention are discussed, and be the most useful and the most intelligible explanation in order to provide anything to be considered to the principle of the invention and design aspect and propose.In this, do not attempt more required for the present invention than basic comprehension to show constructional details of the present invention in greater detail, how several form of the present invention is specialized in practice to accompanying drawing description, those skilled in the art to be understood.
In the accompanying drawings:
Fig. 1 a-g is described in the class bone microenvironment created in the bioreactor system containing 3-D carrier.Fig. 1 a-b be describe nature bone (Fig. 1 a) with inoculating adhering substrate cell (3D-ASC) latter 7 days PluriX tMthe comparison electron micrograph of the imitation bone microenvironment (Fig. 1 b) of 3D carrier structure.Fig. 1 c-f describes the PluriX with the 3D-ASC inoculation produced from marrow tMthe electron micrograph of 3D matrix 20 days after inoculation (Fig. 1 c-d amplifies X150 and 250 respectively) and 40 days (Fig. 1 e-f amplifies X350 and 500 respectively).Fig. 1 g has the Plurix 3D plug flow reactor by the separate part of number definition: post (5), flow monitor (6), flow valve (6a), separation vessel (7), cell growth analysis instrument (8), peristaltic pump (9), sampling spot (10), the molten O of the supply of substratum storehouse (1), mixed gas (2), strainer (3), injection point (4), wherein placement 3D carrier 2potential electrode (11), pH-measuring electrode (12), Controlling System (13), fresh growth medium (14), the growth medium (15) used.
Fig. 2 is described in different production batch (lot) figure (3D-ASC of the adhering substrate cell deriving from placenta grown under 3D growth conditions in bioreactor system; Batch 5-8).By ASC (2X10 6) be inoculated into bio-reactor with the density of 10000-15000 cell/carrier.Cultivate after 12 days, the density of 3D-ASC reaches 150,000-250,000 cell/carrier or containing 150 carriers bio-reactor in 22.5-37.5X10 6individual.。
Fig. 3 a-b is the bar graph of the expression level difference describing the membrane marker (lavender) compared in the membrane marker (mulberry) of expressing in the 3D-ASC being derived from placenta and the placenta cells cultivated under conventional 2D culture condition.Adherent cell is growth 4-6 week in culturing bottle (2D), or in polystyrene support (3D) upper growth 2-3 week in bioreactor system.Make cell from culturing bottle or carrier after results, hatch and with identification MSC (Fig. 3 a) or one group of monoclonal antibody (MAb) of hematopoietic cell (Fig. 3 b) membrane marker feature combine.Notice that MSC membrane marker expression (as shown in for CD90, CD105, CD73 and CD29 membrane marker) in 2D culturing cell is obviously higher than the MSC membrane marker of expressing in the adherent cell cultivated at 3D, especially CD105, in 3D culturing cell, show 56% express, comparing in 2D culturing cell is that 87% (Fig. 3 a).ASC in both 2D and 3D culture does not express any hematopoiesis membrane marker (Fig. 3 b).
Fig. 4 a-d is the bar graph describing the protein level comparing the ASC from placenta generation cultivated under 2D and 3D conditioned medium at 2D and 3D conditioned disjunction.Fig. 4 a-c describes and (is standardized as 1X10 by elisa assay with pg/ml 6individual cell/ml) cultivate in 2D and 3D conditioned medium ASC Flt-3 part (Fig. 4 a), the level of IL-6 (Fig. 4 b) and SCF (Fig. 4 c).Result represents one of three independent experiments.Fig. 4 d shows the expression level of different cell protein, according to the analytical reagent composition with the protein example compared between iTRAQ reagent mark.Protein sample picks up from the ASC grown under 2D (informal voucher) and 3D (lath) condition.This figure represents two of repeating in experiment.Notice the difference of the expression level of some albumen in the conditioned medium of 2D and 3D culture condition and cell.
Fig. 5 a-d describes the Photomicrograph that the 3D-ASC being derived from placenta is divided into osteoblastic ability in vitro.The ASC being derived from Human plactnta cultivates 3 time-of-weeks in the substratum (DMEM containing 10%FCS, 100nM dexamethasone, 0.05mM xitix 2-phosphoric acid salt, 10mM B-glycerophosphate) of leading Osteoblast Differentiation.The cell of Fig. 5 a-b Explicit Expression calcified matrix, according to being dyeed by AlizzarinRed S.Fig. 5 c-d display need not lead the compared with control cells of the medium treatment of Osteoblast Differentiation, and it is held in fibrocyte sample phenotype and proves do not have mineralising.
Fig. 6 describes the chart using chemotherapy (continuous 2 weeks peritoneal injection 25mg/kg busulfans (busulfan)) to treat the people CD45+ cell percentages detected in the NOD-SCID mouse bone marrow cells (BM) of 3.5 weeks after the transfer.CD34+ cell (100,000) from the monocyte purifying being derived from Cord blood is transplanted separately (5 mouse, a), or with the 0.5X10 cultivated under 2D condition 6the individual adherent cell co-transplantation (2D-ASC being derived from placenta; 2 mouse, b), or with at pluriX tMcultivate under 3D condition in bio-reactor be derived from placenta adherent cell (3D-ASC) co-transplantation (5 mouse, c).Then BM is collected from mouse Thigh bone and shin bone.By the people's cell in Flow cytometry BM.The per-cent of people's cell of expressing CD45 is measured by allowing cell and anti-human CD45-FITC hatch.Notice compared with the per-cent (a) of the human cell in alone HSC process mouse, with 2D-ASC (b) and higher with the per-cent of the people's cell (hCD45+) in the mouse bone marrow cells of 3D-ASC (c) co-transplantation.In the mouse through the process of 3D-ASC culturing cell, observe the immigration higher than the mouse through the process of 2D-ASC culturing cell, indicate through 3D cultivate ASC specific to higher treatment advantage.
Fig. 7 a-b only transplants CD45+ cell with the people in the mouse of CD34+ Transplanted cells (Fig. 7 a) adds the facs analysis that the ASC (Fig. 7 b) that is derived from fatty tissue compares with CD34+ cell.Notice compared with (7b-12%) in the mouse of alone people CD34+ process, be derived from fatty tissue ASC co-transplantation mouse in artificial blood colony (hCD45+) per-cent (7a-29%) obviously higher.
Fig. 8 be described in human cord blood monocyte (CB) with etc. radiation quantity (3000Rad) cord blood cell (iCB), be derived from human peripheral blood mononuclear cell (PBMC), 2D cultivates the placenta ASC that (3D) placenta ASC or PBMC and 2D and 3D that (2D) or 3D cultivate cultivate combines the bar graph carrying out mixed lymphocyte reacion between (PBMC+2D and PBMC+3D).CB cell mass size by 3h-thymidine absorbs, and (measuring with CPM) represents, measures in last 18 hours that cultivate.The CB cell proliferation of irriate raises and higher immunne response level is described.Noticing that the cell of hatching with adherent cell demonstrates lower immunne response level, particularly when jointly hatching with adherent cell, the CB immunne response of PBMC being reduced.Each reaction three, work repetition.
preferred embodiment explanation
To be novel methods for cell expansion and the cell produced by this and conditioned medium for stem cell associated treatment, stem cell move into and the purposes of HSC support in the present invention.
Principle of the present invention and operation can be understood better with reference to accompanying drawing and explanation of enclosing.
Before explaining at least one embodiment of the present invention in detail, should be appreciated that the present patent application is not limited to that set forth in following explanation or in embodiment illustration details.The present invention can have other embodiment or practice in every way or implement.Be also to be understood that wording used herein and term are in order to illustration purpose, should not take restriction as.
In growing medical circle, in order to clinical and research purpose more and more need stem cell, stroma stem cell (also referred to as " mescenchymal stem cell ") more specifically.MSC, for supporting HSC to transplant and moving into, is also used for the treatment of more and more various disease conditions, such as heart trouble, BM deficiency disease, neurone relative disease and the illness needing organ or tissue to transplant.
Use the obstacle of stem cell to be to be separated the stem cell of a large amount of normal presence or progenitor cell technically difficult, reason is: these cells limited amount in most tissues, inconvenience and risk is related in the method obtaining stem cell, and with current harvesting method with memory B cell and hemopoietic stem cell loss.Obtain cell from Human embryo on the technical difficulty existed, turn increase problem in religion and ethics.
The alternative source being derived from the stem cell of marrow comprises fatty tissue and placenta.But, there is no effectively amplification at present from the method for the stem cell of these tissues.
The present invention is committed to put into practice time, the present inventor find effectively can breed under 3D culture condition from the adherent cell of placenta or fatty tissue.The present inventor finds that such cell comprises the functional performance similar to MSC unexpectedly, and therefore, these cells and consequent conditioned medium can be used as therapeutic purpose, and such as, in transplanting, tissue regeneration and body, HSC supports.
As hereafter with as described in following examples part, the present inventor can increase and comprise the adherent cell being derived from fatty tissue and placenta of stroma stem cell characteristic in 3D environment.Find that the cell increased accordingly has vigor after low temperature storage, this proves (see embodiment 1) by adhering to and heavily formed colony's mensuration (repopulation assay).The flow cytometry being derived from the adherent cell of placenta finds different marker expression patterns (see Fig. 3 a-b).The most important thing is, the adherent cell being derived from fatty tissue and placenta of breeding in 2D or 3D environment can support that HSC moves into (see embodiment 2), which confirms cell of the present invention as stroma stem cell purposes clinically.
Therefore, one aspect of the present invention provides the method for cell amplification.
Described method cultivates the adherent cell from placenta or fatty tissue under being included in three-dimensional (3D) culture condition of sustenticular cell amplification.
Term used herein " amplification " and " amplification " refer to maintain cell and final cell growth, even if cell mass increases (such as at least to 2 times) and there is not the differentiation with described increase substantially undifferentiatedly.
Term used herein " maintenance " and " maintenance " refer to substantially undifferentiated cell turnover, i.e. stabilized cell group and do not exist with described stable differentiation substantially.
Phrase used herein " adherent cell " refers to homogeneity or the foreign cell group with anchorage dependence, and namely cell is in order to growth needs adhesive surface in vitro.
Phrase used herein " fatty tissue " refers to the reticular tissue comprising adipocyte (adipocyte).
Term used herein " placenta tissue " refers to along Uterus wall and wraps up any part of the female organ of fetus at gestation time, and placenta is connected by umbilical cord with fetus.Ablatio placentae (being called postpartum placenta) after birth.
Phrase used herein " three-dimensional cultivation condition " refers to cell to be placed in Growth of Cells that the compatible cell that simultaneously makes can more than the condition that one deck grows.The in situ environment of cell in live organism (or tissue) can be interpreted as three-dimensional structure completely.Cell by other cell around.They are fixed in the composite network of the nanoscale cell epimatrix fiber making to set up various local microenvironment.Their the outer part of born of the same parents not only mediates the attachment to basilar membrane, but also leads to multiple blood vessel and lymphatic vessel.Oxygen, hormone and nutrient substance are transported to cell refuse and are then transported.Three-dimensional cultivation condition of the present invention is designed to simulate the such as following environment being further used as illustration.
Therefore, the adherent cell of this respect of the present invention extracts from fatty tissue or placenta tissue.
Placenta cells can be obtained from mature or preterm labor placenta.Preferably just collect placenta once external hemorrhage.Preferably within the time being enough to removing residual cell, pour into placenta.Term used herein " perfusion (perfuse or perfusion) " refers to be poured into by liquid or make liquid by the behavior of organ or tissue.Placenta tissue can from any Mammals; Most preferred placenta tissue is the placenta tissue of people.It is postpartum placenta (such as 1-6 hour) that placenta tissue is originated easily, but, the source of placenta tissue or cell or to be separated the method for placenta tissue unimportant for the present invention.
The adherent cell being derived from placenta can from the fetus of placenta (i.e. amnion or placenta internal portion, see embodiment 1) and parent (i.e. basal decidua and parietal decidua) part.Washing tissue sample in normal saline buffer solution [such as phosphate buffered saline (PBS) (PBS) or Hank damping fluid).By with digestive ferment process tissue (see below) or/and chopping (mince) and with washing substratum through NF rinse tissue part or by gently piping and druming (Falcon, Becton, Dickinson, San Jose, CA), single-cell suspension liquid is prepared.
The adherent cell being derived from fatty tissue is separated by multiple method well known by persons skilled in the art.Such as, such method is set forth in United States Patent (USP) the 6th, 153, No. 432.Fatty tissue can be derived from nethike embrane/internal organ, mammary gland, sexual gland or other adipose tissue site.Preferred fat is tissue-derived is omental adipose tissue.In the mankind, fatty tissue is separated by liposuction usually.
Adherent cell is separated by obtaining at following condition process tissue: with digestive ferment (such as collagenase, trypsinase and/or Dispase from fatty tissue; And/or the Unidasa of effective concentration or DNA enzymatic); With ethylenediamine tetraacetic acid (EDTA) (EDTA); At the temperature action 10 minutes-3 hours of 25-50 DEG C.Then cell can be allowed by the nylon of 20 microns-800 microns or cheese cloth net filter.Then cell is allowed directly to carry out differential centrifugation in the medium or through Ficoll or Percoll or other particle gradient.Cell 4-50 DEG C of temperature with 100-3000xg centrifugation 1 minute-1 hour (see United States Patent (USP) the 7th, 078, No. 230).
Except being derived from the adherent cell of placenta or fatty tissue, the present invention also looks forward to the purposes of the adherent cell from other cell derived being characterized as stromal stem cell phenotype (it will hereafter set forth further).Can include but not limited to from wherein extracting the tissue-derived of adherent cell: Cord blood, hair follicle [such as described in No. 20060172304th, U.S. Patent application], testis [such as Guan K. etc., Nature.2006 Apr 27; Described in 440 (7088): 1199-203], people's olfactory mucosa [such as Marshall, CT. etc., Histol Histopathol.2006 Jun:21 (6): described in 633-43], embryonic yolk sac [such as Geijsen N, Nature.2004 Jan 8; Described in 427 (6970): 148-54] and amniotic fluid [Pieternella etc. (2004) Stem Cells.22:1338-1345], known they all comprise mescenchymal stem cell.By making these cells culture of isolated on the adhesive surface, adherent cell can be isolated thus from other cell primitive horde from these tissue-derived adherent cells.
No matter be any source (such as placenta or fatty tissue), preferably aseptically extract cell.Once obtain the cell be separated, just make it adhere on adhesion material (such as forming surface), be separated adherent cell by this.This can be to cultivate in 3D culture condition before (see embodiment 1) or carry out simultaneously.
" adhesion material " used herein refers to have can allow cell remain on the synthetic of the chemical structure (such as lotus has surface exposed groups) on its surface, natural existence or its nontoxic (i.e. physiologically acceptable) material combined.
The adhesion material example that can be used for this respect of the present invention includes but not limited to: polyester, polyalkenes, poly-fluorine vinylchlorid, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibre, ceramic particle, matrigel, extracellular matrix components (such as fibronectin splicing variants, chondronectin, ln), collagen, poly-L-lactic acid and inert metal fiber.
Can realize being further purified by well known method or the step (such as by the FACS with stroma stem cell marker expression, it is hereafter being set forth further) of enriched medium stem cell.
Limiting examples for the minimum medium of the present invention's cultivation comprises the necessary substratum Eagle of the limit, ADC-1, LPM (without bovine serum albumin), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI1640, BGJ substratum (carry out and do not carry out Fitton-Jackson improvement), minimum medium Eagle (BME-adds Earle alkali salt), Dulbecco improves Eagle substratum (DMEM-serum-free), Yamane, IMEM-20, Glasgow improves Eagle substratum (GMEM), Leibovitz L-15 substratum, McCoy5A substratum, substratum M199 (M199E-is containing Earle alkali salt), substratum M199 (M199H-is containing Hank alkali salt), the necessary substratum Eagle of the limit (MEM-E-is containing Earle alkali salt), the necessary substratum Eagle of the limit (MEM-H-is containing Hank alkali salt) and the necessary substratum Eagle (MEM-NAA, containing nonessential amino acid) of the limit, comprises at other substratum numerous: substratum 199, CMRL1415, CMRL1969, CMRL1066, NCTC135, MB 75261, MAB 8713, DM 145, Williams ' G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153.Be DMEM for preferred culture medium of the present invention.These and other useful substratum purchased from GIBCO, Grand Island, N.Y., USA and Biological Industries, Bet HaEmek, Israel etc.These substratum a large amount of are summarized in Enzymology method (Methods inEnzymology) LVIII volume, " cell cultures (Cell Culture) ", 6272nd page, WilliamB.Jakoby and Ira H.Pastan edits, and Academic publishing company publishes.
Substratum can supplement such as serum (the tire serum of such as ox or other species or) and optional or alternative with the somatomedin of pik/ml-milligram/ml level concentration, cytokine and hormone (such as tethelin, erythropoietin, thrombopoietin, interleukin-13, interleukin 6, IL-7, macrophage colony stimulating factor, c-kit part/STEM CELL FACTOR, osteoprotegerin ligand, Regular Insulin, rhIGF-1, Urogastron, fibroblast growth factor, nerve growth factor, ciliary neurotrophic factor, platelet derived growth factor and bone morphogenetic protein).
Should recognize further and other component can be added in substratum.Such component can be microbiotic, antifongin, albumin, amino acid and other component for cell cultures known in the art.In addition, component can be added when needing to improve atomization (see following further elaboration).
Three-dimensional environment (embodiment 1 see following examples part) just can be transferred to once obtain adherent cell.But should understand, immediately described cell can be transferred to 3D after isolation and construct in matrix (as described above).
Therefore, the adhesion material of this respect of the present invention is shaped for 3D cultivation, provides the growth matrix substantially increasing and stroma cell is stained with to use attaching surface by this, so that the foundation structure of simulated tissue (such as placenta).
Such as, for the growth matrix that 0.5mm is high, doubly, it is by projecting to growth matrix base portion to calculate to be increased at least 5-30.The described about 5-30 that is increased to is doubly for each individual layer, if use many described layers (no matter be stacking or separated by spacer or like this), then 5-30 is doubly applicable to every one deck structure like this.When matrix uses with sheet (sheet) form, preferred non-woven fiber sheet or hole-opening foaming polymer sheet, preferred sheet thickness is about 50-1000 μm or thicker, its provide enter for cell, nutrient substance enters and removes enough porousness of refuse from sheet.According to preferred embodiment, described hole has the effective diameter of 10 μm-100 μm.Such sheet can be prepared from the fiber of various thickness, and optimum fiber thickness or fiber diameter range are about 0.5 μm-20 μm, and more preferably fiber diameter range is 10 μm-15 μm.
Structure of the present invention can be supported by the open support sheet or screen cloth providing spatial stability and physical strength, or even better and its combination.
Also can cut, punching or shred such substrate tablet, be about 0.2mm-to provide shadow area and be about 10mm and the particle of about same thickness (about 50-1000 μm).
Manyly be set forth in United States Patent (USP) the 5th with for making the present invention be committed to the making of the growth matrix of practice, purposes and/or advantage, 168, No. 085 and particularly in the 5th, 266, No. 476, they are all hereby incorporated by reference.
Adhesive surface can have and is selected from square, annular, circle and criss-cross shape.
For scale operation, preferably realize cultivating in 3D bio-reactor.
Such thing reactor example includes but not limited to plug flow reactor, continuously stirring groove tank bio-reactor and fixed-bed bioreactor.
As shown in the embodiment 1 of embodiment part, three-dimensional (3D) plug flow reactor (as described in No. 6911201st, United States Patent (USP)) can the growth of supported matrix cell and long term maintenance.In this bio-reactor, stroma cell is inoculated on the porrosive carrier that is loaded on and is made up of non-woven fiber polyester matrix in glass column, makes a large amount of cell breed in relatively little volume by this.
Matrix used in plug flow reactor can be sheet form, for non-woven fiber sheet or hole-opening foaming polymer sheet, preferred sheet thickness is about 50-1000 μm or thicker, its provide enter for cell, nutrient substance enters and removes enough porousness of refuse from sheet.
Other 3D bio-reactor that can use with the present invention includes but not limited to that continuous agitator tank bio-reactor (wherein makes substratum be continuously fed in bio-reactor, extracted out continuously by product, stable state time permanent to maintain in reactor.Stirred-tank bioreactor with fibre bed basket can purchased from such as New Brunswick Scientific company Edison, NJ), fixed-bed bioreactor, (wherein air passes into the bottom of center conduit to airlift bioreactor usually, upwards flow and form bubble simultaneously, waste gas is separated) at capital, cell with Polyactive porous plastics inoculates filling type bioreactor (as Wendt, D. etc., Biotechnol Bioeng 84:205-214, (2003) described), tubulose poly-L-lactic acid (PLLA) porous support radial flow filling type bioreactor is [as Kitagawa etc., Biotechnology and Bioengineering 93 (5): 947-954 (2006) is described].Other bio-reactor according to the invention is set forth in United States Patent (USP) the 6th, 277, No. 151, the 197th, No. 575, the 139th, No. 578, the 132nd, No. 463, the 902nd, No. 741 and the 5th, 629, No. 186.
During inoculation, preferred cell inoculation reaches 100,000-1,500,000 cell/mm.
Preferably once reach at least about 40% converging, 60% converge or 80% converge with regard to harvested cell, preferably avoid uncontrolled differentiation and aging simultaneously.
Cultivation is carried out at least about 2 days, 3 days, 5 days, 10 days, 20 days, 1 month or even longer.Should understand, the cultivation in bio-reactor may extend this time.May realize going down to posterity to increase cell number.
Preferred adherent cell of the present invention comprises at least one " stromal stem cell phenotype ".
" stromal stem cell phenotype " used herein refers to the typical structure or the function phenotype that are derived from bone marrow matrix (i.e. mesenchyme) stem cell.
Phrase used herein " stem cell " refers to the cell not having end differentiation eventually.
Therefore, such as, cell may have fusiform.As alternative or in addition, cell can express one or a group (such as surface markers) mark of stroma stem cell symbol.Stroma stem cell surface markers (positive and negative) example includes but not limited to CD105+, CD29+, CD44+, CD73+, CD90+, CD34-, CD45-, CD80-, CD19-, CD5-, CD20-, CD11B-, CD14-, CD19-, CD79-, HLA-DR-and FMC7-.Other stroma stem cell mark includes but not limited to tyrosine hydroxylase, nidogen and H-NF.
Stroma stem cell representative functions phenotype example includes but not limited to T cell inhibit activities (do not stimulate T cell and suppress it on the contrary), hemopoietic stem cell supports active and adipocyte, liver cell, scleroblast and neurocyte directed differentiation.
Any one in these structures or functional character all can be used for evaluation cell of the present invention (the embodiment 1-2 see following examples part).
Instruct the cell mass of generation to be characterized as unique protein expression profile according to the present invention, it is as shown in the embodiment 1 of embodiment part.Therefore, such as, the placenta of generation or fatty tissue adherent cell is instructed to express according to the present invention and/or the factor selected by secreting high levels.Such as, such cell expressing or secretion SCF, Flt-3, H2AF or ALDH X than growth in cultivating at 2D placenta fatty tissue adherent cell is expressed or secretion up at least 2,3,4,5,6,7,8,9,10,11 or preferably 12 times.In addition or as alternative, cell population secretes of the present invention or express IL-6, EEEF2, RCN2 or CNN1 than growth in cultivating at 2D placenta fatty tissue adherent cell is expressed or secretion up at least 2,3 or 5 times of levels.In addition or as alternative, it is lower that cell mass of the present invention is characterized as other protein expression level more various with 2D cultured cells.Therefore, such as, secretion or expression ratio 2D cultivate in growth placenta fatty tissue adherent cell express or secretion Hnrph1, CD44 isotype 2 precursor, Papss2 or rpL7a expression level be low to moderate 0.6,0.5,0.25 or 0.125.
When the present invention being specialized further in practice, the present inventor recognizes adhering substrate cell especially 3D-ASC display immunosuppressive activity.As shown in the embodiment 3 of following examples part, find in MLR measures adhering substrate cell especially 3D-ASC suppress the immune response of Human Umbilical Cord blood monocyte.Therefore, cell of the present invention can comprise can preferentially for clinical biological activity (such as support active) by T cell inhibit activities, hemopoietic stem cell.
When the present invention being specialized further in practice, the present inventor recognizes that the conditioned medium of cell of the present invention can comprise can preferentially for clinical biological activity (such as T cell inhibit activities, hemopoietic stem cell support activity).
Therefore, the present invention look forward to further the collection of conditioned medium and its same as before or application concentrating by well known method, after the other step of enrichment or fractionation.Preferred conditioned medium of the present invention is from the cell cultures mid-log phase of high viability.
As mentioned above, cell of the present invention and conditioned medium are characterized as stromal stem cell phenotype, therefore can be used for any research and may benefit from any clinical application of the such cell of use.
The HSC transplanted moves into and starts hemopoietic and depends on complicated process, comprise and to go back to the nest marrow reside in suitable tabernacle through endothelial cell barrier with chemokine gradient, the transplanted cells simultaneously in tabernacle, between ECM and mesenchymal cell, set up physical contact.All these processes relate to the molecule of a group complexity, such as cytokine, hormone, steroid, extracellular matrix protein, somatomedin, iuntercellular effect and adhesion protein and stromatin.
In receptor BM, HSC [Kerre etc., the J Immunol.167:3692-8. (2001) that 1-5% inculcates within after known transplanting 2-3 days, only detected; Jetmore etc., Blood.99:1585-93 (2002)].
MSC is to suppress the T cell being derived from donor causing graft versus host disease (GVH disease) to produce [(GvHD, Charbord P., and Moore, K., Ann.N.Y.Acad.ScI 1044:159-167 (2005) to the contribution part that hematopoiesis moves into; Maitra B etc., Bone Marrow Transplant.33 (6): 597-604. (2004); United States Patent (USP) the 6th, 010, No. 696; No. 6555374], part is to provide hemopoietic stem cell (HSC) to support (namely maintain and help hemopoietic stem cell proliferation, maturation and/or go back to the nest).
As described in the embodiment 2 of following examples part, even if unexpectedly find that the adherent cell being derived from placenta and fatty tissue also supports that after chemotherapy HSC moves into.
Consider these results, can imagine that cell of the present invention or substratum can be used in any clinical application using stroma stem cell to transplant.
Therefore, the present invention is provided on the other hand needs treatment in the object for the treatment of can benefit from the method for the medical conditions (such as pathology, disease, syndrome) that stroma stem cell is transplanted.
Term used herein " treatment " refers to suppress or block pathological evolution and/or cause alleviating, eliminate or disappearing of pathology.It should be appreciated by those skilled in the art that can use various method and measure and assess pathological evolution, similarly, various method can be used and measure and assess alleviating, eliminate or disappearing of pathology.Term " treatment " preferably refers to slow down or reduce the symptom relevant with carninomatosis.Preferred therapeutic cures the symptom that such as elimination is relevant with medical conditions substantially.
" can benefit from the medical conditions that stroma stem cell is transplanted " used herein refers to any medical conditions slowed down by giving cell/substratum of the present invention.
Term or phrase " transplanting ", " cell exchange " or " immigration " can be used alternatingly in this article, refer to cell of the present invention to be incorporated in target tissue.
Term used herein " object " refers to any object (such as Mammals), preferred human subjects.
The method of this respect of the present invention comprises the cell of the present invention or substratum (above-mentioned) that give described subject significant quantity, the medical conditions of the stroma stem cell the benefited from transplanting for the treatment of target by this.
The cell that can give according to this respect of the present invention comprises the above-mentioned adherent cell that can cultivate in two dimension or three-dimensional environment and mesenchyme thereof and non-mesochymal part or end differentiation derivative eventually.
The derivative method from the particular lineage cell of stroma stem cell of the present invention is well known.See such as No. the 5th, 486,359, United States Patent (USP), the 5th, 942, No. 225, the 5th, 736, No. 396, the 5th, 908, No. 784 and the 5th, 902, No. 741.
Cell can be natural, or genetic improvement is such as so that derivative object pedigree (see No. 20030219423rd, U.S. Patent application).
Cell and substratum can be autologous or non-autologous (i.e. allogeneic or xenogenesis) prepared by fresh or freezing (such as freezen protective).
The other chemicals of described object (such as immunomodulator, chemotherapy etc.) or cell can be given according to medical conditions.
Therefore, move into (such as have the HSC number of viability and the optimization increased in receptor BM improves normal white blood cell count) to improve stem cell, such as can by cell/substratum of the present invention before HSC transplants, simultaneously or give afterwards.
Preferred HSC and stroma cell enjoy common HLA antigen.Preferred HSC and stroma cell are from single individuality.Or HSC and stroma cell are from Different Individual.
Term or phrase " transplanting ", " cell exchange " or " immigration " can be used alternatingly in this article, refer to cell of the present invention to be incorporated in target tissue.Described cell can be derived from receptor or be derived from allogeneic or xenogenesis donor.
Because may react by induction of immunity when non-autogenous cell being given health, repel the somatic possibility of non-self so developed several method to reduce.These methods comprise and suppress receptor's immunity system before transplantation or be coated on by non-autogenous cell in immunity isolation (immunoisolating) semipermeable partition.
Encapsulated (encapsulation) technology is divided into the microcapsule relating to little spheroid carrier and large capsule (the macroencapsulation) (Uludag relating to larger dull and stereotyped hollow-fibre membrane usually, H. etc., mammalian cell encapsulation techniques (Technology of mammalian cellencapsulation) .Adv Drug Deliv Rev.2000; 42:29-64).
Prepare the method for microcapsule for described in this area, comprise such as following disclosed method: LuMZ etc., cell encapsulation (Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly (allylamine)) .Biotechnol Bioeng.2000, the 70:479-83 of (allylamine) is gathered with alginate and α-phenoxy group cinnamylidene acetylize; Chang TM and Prakash S, microencapsulation method (the Procedures for microencapsulation of enzymes of enzyme, cell and genetically engineered microorganism, cells and genetically engineered microorganisms) .Mol Biotechnol.2001,17:249-60; With Lu MZ etc., by the method (A novel cell encapsulation method usingphotosensitive poly (allylamine alpha-cyanocinnamylideneacetate)) of the novelty of photosensitive poly-(allylamine alpha-cyano cinnamylidene acetic ester) encapsulated cell.JMicroencapsul.2000，17：245-51。
Such as, preparing microcapsule by being mixed with HEMA (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA) ter-polymers shell by the collagen of improvement, causing the thick 2-5 μm of capsule.Such microcapsule can further with other 2-5 μm of ter-polymers shell bag quilt, (Chia is minimized to provide the smooth surface of negative charge and to make plasma proteins absorb, S.M. etc., as Multi-layer microcapsule (the Multi-layered microcapsules for cell encapsulation Biomaterials) .200223:849-56 of cell encapsulation biomaterial).
Other microcapsule are based on alginate, marine polysaccharide (Sambanis, A, encapsulated pancreas islet (Encapsulated islets in diabetes treatment) .Technol.Ther.2003,5:665-8 in treating diabetes) or derivatives thereof.Such as, microcapsule are prepared by polyanion sodiun alginate and the polyelectrolyte complexing action between Ushercell and polycation methylene radical-guanidine copolymer salt hydrochlorate under calcium chloride existence.
Should understanding, improving cell encapsulation when using less capsule.Therefore, when capsule size is reduced to 400 μm from 1mm, the quality control of encapsulated cell, mechanical stability, dispersing characteristic and external activity are all improved (Canaple L. etc., control to improve cell encapsulation (lmproving cell encapsulation through size control) .JBiomater SciPolym by size to edit, 2002; 13:783-96).In addition, find that aperture controls well at the little nanoporous biological capsule to 7nm, treated (tailored) surface chemistry and accurate micro-structure that successfully (namely Williams D. is little is U.S.: the particulate in medical treatment device and nanoparticle technology (Small is beautiful:microparticle andnanoparticle technology in medical devices) .Med Device Technol.1999,10:6-9 for cellular immunization isolates microenvironment; The encapsulated microstructuring techniques of Desai, T.A. pancreatic cell (Microfabricationtechnology for pancreatic cell encapsulation) .Expert Opin Biol Ther.2002,2:633-46).
Immunosuppressor example includes but not limited to: methotrexate (methotrexate), endoxan (cyclophosphamide), ciclosporin (cyclosporine), Ciclosporin A (cyclosporin A), chloroquine (chloroquine), Oxychloroquine, sulfasalazine (sulphasalazopyrine), gold salt, Beracilline (D-penicillamine), leflunomide (leflunomide), azathioprine (azathioprine), Kineret (anakinra), infliximab (infliximab) (REMICADE), etanercept (etanercept), TNF alpha blocker, the bio-pharmaceutical of target inflammatory cytokine, with nonsteroidal anti-inflammatory drugs (NSAID).NSAID example includes but not limited to: acetylsalicylic acid, choline salicylate magnesium, diflunisal (diflunisal), magnesium salicylate, sasapyrin, sodium salicylate, diclofenac (diclofenac), R-ETODOLAC, fenoprofen (fenoprofen), flurbiprofen (flurbiprofen), indomethacin (indomethacin), Ketoprofen (ketoprofen), ketorolac, meclofenamic acid ester, Naproxen Base (naproxen), nabumetone (nabumetone), Phenylbutazone (phenylbutazone), piroxicam (piroxicam), sulindac, tolmetin (tolmetin), paracetamol (acetaminophen), Ibuprofen BP/EP (ibuprofen), Cox-2 inhibitor and U-26225A (tramadol).
In either method described herein, cell or substratum itself can be given, or preferably give as the pharmaceutical composition part comprising pharmaceutically acceptable carrier further.
" pharmaceutical composition " used herein refers to the preparation of one or more the chemically conjugated things described herein containing other chemical composition (such as pharmaceutically suitable carrier and vehicle).Pharmaceutical composition object conveniently gives object by compound.
Hereafter term used " pharmaceutically acceptable carrier " refers to carrier or thinner, and it does not cause obvious stimulation to object, does not eliminate biologic activity and the characteristic of given compound.The limiting examples of carrier is the mixture of propylene glycol, salt solution, emulsion and organic solvent and water.
Term used herein " vehicle " refers to be added in pharmaceutical composition to further facilitate the inert substance of administered compound.The limiting examples of vehicle comprises calcium carbonate, calcium phosphate, various sugar and all kinds starch, derivatived cellulose, gelatin, vegetables oil and polyoxyethylene glycol.
According to the preferred embodiment of the invention, pharmaceutical carriers is aqueous saline solution.
Preparation and give the technology of medicine can at " Lei Mingdun pharmaceutical science (Remington ' sPharmaceutical Sciences) ", Mack publishing company, Easton, PA, find in latest edition, and it is hereby incorporated by reference.
Systemic fashion can give pharmaceutical composition (as described above).Or, locally can give pharmaceutical composition, such as direct pharmaceutical composition is expelled to patient organize district.
Prepare pharmaceutical composition of the present invention by method well known in the art, such as by means of routine mixing, dissolving, granulating, make dragee, levigation, emulsification, encapsulated, embedding or the method for freeze-drying.
Therefore, on the physiology that can comprise vehicle and auxiliary with one or more, acceptable carrier is allocated in a usual manner for pharmaceutical composition of the present invention, and described carrier is convenient is processed into the preparation that can pharmaceutically use by activeconstituents.Suitable preparation depends on selected route of administration.
For injection, the activeconstituents of pharmaceutical composition can be allocated with the aqueous solution, preferably with the damping fluid that physiology can hold such as Hank solution, Ringer solution or normal saline buffer solution.For for mucosa delivery, in preparation, use the permeate agent being suitable for barrier to be infiltrated.Such permeate agent is generally known in the art.
For for any preparation in the inventive method, from external and cell cultures measure, initial estimation significant quantity or dosage can be treated.Preferably in animal model allocating dosage to reach required concentration or titre.Such data can be used for the dosage determining that the mankind use more accurately.
In cell cultures or laboratory animal, toxicity and the curative effect of activeconstituents described herein is measured by Standard in vitro method of pharmacy.
Can be used for allocating a series of dosage for the mankind from these external data measured with cell cultures and obtain zooscopy.The visual formulation used of dosage and route of administration used and change.Consider that patient condition selects definite preparation, route of administration and dosage (see such as Fingl etc. by individual physician, 1975, be loaded in " therapeutic pharmacological basis (ThePharmacological Basis of Therapeutics) ", the 1st chapter page 1).Such as, symptom monitoring can be carried out to the motor function of the display of disturbances in patients with Parkinson disease to treatment active responding.
For injection, the activeconstituents of pharmaceutical composition can be allocated with the aqueous solution, preferably with the damping fluid that physiology can hold such as Hank solution, Ringer solution or normal saline buffer solution.
Dosage and dosing interval can being adjusted individually, effectively regulating neurotransmitter to synthesize with the cell making active ingredient level be enough to by transplanting.The dosage reached needed for ideal effect will depending on personal feature and route of administration.Detection experiment can be used for measuring plasma concentration.
According to severity and the reaction of illness to be treated, can single or multiple administration, and continue some skies the course for the treatment of to some weeks, or reach the state of palliating a disease.
Certainly, the amount of composition to be administrated is by the judgement etc. depending on just meeting subject individuality, ailing severity, administering mode, doctor in charge.Dosage and time should make a response to the individual change of illness state of carefully monitoring continuously.Such as, based on the instruction of monitoring, the cell concentration of the symptom that the disturbances in patients with Parkinson disease be treated is enough to palliate a disease.
Cell of the present invention is preferably survived for some time (such as at least 6 months), to observe result for the treatment of in affected areas after transplanting.
Comprising allotment can through preparation at interior composition at the invention formulation that medicine can hold in carrier; Be placed in suitable vessel; And put on the treatment being used to specify illness.
If necessary, the present composition can be stored in bag or distribution device (dipenser device) test kit that such as FDA checks and approves, and they can containing the one or more unit dosage containing activeconstituents.Described bag can such as comprise metal or plastic sheet, the bag that such as bubbles (blister pack).Described bag or distribution device can have administration explanation.Bag or distribution device also can provide with by the relevant bulletin of the container of form of governmental agency requirements managing drug manufacture, use or sale, described bulletin reflects the composition forms that this mechanism ratifies or mankind's administration or veterinary administration form.Such bulletin can be such as by U.S. food and the prescription drug label of drugs administration approved or the product inset of approval.
Embodiment
Present following examples form reference with illustrating in a non-limiting manner together with above-mentioned explanation of the present invention.
Term used herein and the present invention experimentally generally include molecule, biochemistry, microbiology and recombinant DNA technology.These technology have detailed explanation in the literature.See such as: " molecular cloning: laboratory manual (Molecular Cloning:A laboratory Manual) " Sambrook etc., (1989); " molecular biology general scheme (Current Protocols inMolecular Biology) " I-III rolls up, and Ausubel, R.M. edit (1994); Ausubel etc., " molecular biology general scheme (Current Protocols in Molecular Biology) ", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, " molecular cloning practical guide (A Practical Guide to Molecular Cloning) ", John Wiley & Sons, NewYork (1988); Watson etc., " recombinant DNA (Recombinant DNA) ", ScientificAmerican Books, New York; Birren etc. (editor) " genome molecules: laboratory manual book series (Genome Analysis:A Laboratory Manual Series) ", 1-4 rolls up, ColdSpring Harbor Laboratory Press, New York (1998); United States Patent (USP) the 4th, 666, No. 828, the 4th, 683, No. 202, the 4th, 801, No. 531, the 5th, 192, No. 659 and the 5th, 272, No. 057 method proposed; " cytobiology: laboratory manual (Cell Biology:ALaboratory Handbook) ", I-III rolls up Cellis, J.E. and edits (1994); " immunology general scheme (Current Protocols in Immunology) " I-III rolls up Coligan J.E. and edits (1994); Stites etc. (editor), " basis and clinical immunology (Basic and ClinicalImmunology) " (the 8th edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (editor), " cellular immunology system of selection (Selected Methods in CellularImmunology) ", W.H.Freeman and Co., New York (1980); Extensively be set forth in the useful immunoassay in patent and scientific literature, see such as No. the 3rd, 791,932, United States Patent (USP), the 3rd, 839, No. 153, the 3rd, 850, No. 752, the 3rd, 850, No. 578, the 3rd, 853, No. 987, the 3rd, 867, No. 517, the 3rd, 879, No. 262, the 3rd, 901, No. 654, the 3rd, 935, No. 074, the 3rd, 984, No. 533, the 3rd, 996, No. 345, the 4th, 034, No. 074, the 4th, 098, No. 876, the 4th, 879, No. 219, the 5th, 011, No. 771 and the 5th, 281, No. 521; " oligonucleotide synthesis (Oligonucleotide Synthesis) " Gait, M.J. edits (1984); " nucleic acid hybridization (Nucleic Acid Hybridization) " Hames, B.D., and Higgins S.J. edits (1985); " transcribe and translate (Transcription and Translation) " Hames, B.D., and Higgins S.J. edits (1984); " animal cell culture (Animal Cell Culture) " Freshney, R.L edits (1986); " immobilized cell and enzyme (Immobilized Cells and Enzymes) " IRLPress, (1986); " molecular cloning carries out guide (A Practical Guide to MolecularCloning) " Perbal, B., (1984) and " Enzymology method (Methods in Enzymology) " 1-317 roll up, Academic Press; " PCR scheme: methods and applications guide (PCR Protocols:A Guide To Methods and Applications) ", Academic Press, San Diego, CA (1990); Marshak etc., " protein purification and qualification strategy-laboratory handbook (Strategiesfor Protein Purification and Characterization-A Laboratory CourseManual) " CSHL Press (1996); Allly above to be hereby incorporated by reference as proposed completely herein.Run through the literature and provide other general bibliography.Think that method is wherein well known, provide them to be conveniently readers.Wherein comprised all data are hereby incorporated by reference.
Embodiment 1
Produce from marrow, placenta and fatty tissue and cultivate adhering substrate cell (ASC)
Adherent cell is cultivated, to produce the 3D-ASC cell being characterized as specific cells marker expression spectrum in the bioreactor system containing 3D carrier.By cell counting measuring growth efficiency.By cultivating the differentiation capability measuring these cells in division culture medium.
Material and test method
Marrow stromal cell-obtain marrow (BM) stroma cell from the donor sucking-off bone marrow of sternum of hematology health through cardiac operation under direct vision or BM biopsy.With Hank balanced salt solution (HBSS; GIBCO BRL/Invitrogen, Gaithersburg MD) Bone marrow aspirates is diluted 3 times, carry out Ficoll-Hypaque (Robbins Scientific company, Sunnyvale, CA) density gradient centrifugation.After this collect myelomonocyte (<1.077gm/cm3), wash 3 times with HBSS, Eddy diffusion [supplements 10%FCS (GIBCO BRL), 10 in growth medium -4m mercaptoethanol (Merck, White House Station, NJ), Pen .-Strep-nystatin mixture (100U/ml:100ug/ml:1.25un/ml; Beit Ha ' Emek), the DMEM (Biological Industries, Beit Ha ' emek, Israel) of 2mM L-glutaminate (Beit Ha ' Emek)].In 37 DEG C of (5%CO in tissue culture culturing bottle (Corning, Acton, MA) 2) separately hatch cell from each donor and replaced medium weekly.Within every 3-4 days, divide (split) cell with 0.25% trypsinase-EDTA (Beit Ha ' Emek).After passing 2-40 generation, when reaching 60-80% and converging, collecting cell is for analysis or for cultivating in bio-reactor.
Be derived from the inside part (Bnei Zion medical center, Haifa, Israel) of the stroma cell of placenta-aseptically cut term birth placenta, with Hank buffer solution 3 times, in 37 DEG C, (1mg/ml organizes with 0.1% collagenase; Sigma-Aldrich, St.Lewis, MO) hatch 3 hours.Blow and beat gently with suction pipe, then with the cell that the DMEM washing supplementing 10%FCS, Pen .-Strep-nystatin mixture (100U/ml:100ug/ml:1.25un/ml) and 2mM L-glutaminate suspends, be inoculated into 75cm 2in culturing bottle and in 37 DEG C at 5%CO 2hatch in tissue culture incubator under humidification conditions.After this make cell at frosting adherent 72 hours, then often within 3-4 days, change a subculture.When reaching 60-80% and converging (usual 10-12 days), with 0.25% trypsinase-EDTA from grown cultures bottle isolated cell, and be inoculated in new culturing bottle.After this cultured cells is collected for analysis or for cultivating in bio-reactor.
Be derived from the stroma cell of fatty tissue-obtain stroma cell (Rambam Haifa, Israel) from the human fat tissue of liposuction method.Thoroughly fatty tissue is washed, with collagenase (20mg/ml) in 37 DEG C of digestion 30 minutes with isopyknic PBS.Then with the DMEM washed cell containing 10%FCS, Pen .-Strep-nystatin mixture (100 U/ml:100ug/ml:1.25un/ml) and L-glutaminate, with 1200rpm in room temperature centrifugal 10 minutes, with lysate Eddy diffusion (1:10; Biological Industries, Beit Ha ' emek, Israel, to discard hemocyte), centrifugal, with the DMEM Eddy diffusion containing 10%FCS, Pen .-Strep-nystatin mixture (100U/ml:100ug/ml:1.25un/ml) and L-glutaminate.Then with 3-10x10 7washed cell is inoculated into sterile tissue culture media culturing bottle by individual cell/culturing bottle.Second day with PBS washed cell to remove residual RBC and dead cell.In 37 DEG C at 5%CO 2in tissue culture incubator, cell is kept under humidification conditions.Every 3-4 days replaced medium.When 60-80% converges, with 0.25% trypsinase-EDTA from grown cultures bottle isolated cell, and be inoculated in new culturing bottle.After passing 2-40 generation, when reaching 60-80% and converging, collecting cell is for analysis or for cultivating in bio-reactor.
PluriX tMplug flow reactor-load PluriX with the 3D porrosive carrier (diameter 4mm) that the 1-100ml be made up of non-woven fiber polyester matrix wraps tMplug flow reactor (Pluristem, Haifa, Israel; As described in Fig. 1 g, also see United States Patent (USP) the 6th, 911, No. 201).These carriers make to breed a large amount of cell count in relatively little volume.Designed by Pluristem and manufacture glassware.Bio-reactor keeps in 37 DEG C of incubators, and flow velocity is regulated by valve (6a in Fig. 1 g) and peristaltic pump (in Fig. 1 g 9) and monitored.Bio-reactor contains sampling and injection point (in Fig. 1 g 4), and making can vaccinization cell.The substratum of supply pH 6.7-7.4 from storehouse (Fig. 1 g 1).This storehouse is by the air/CO containing different ratios 2/ O 2the mixture of filtering gas (in Fig. 1 g 2, the 3) supply of (being determined by the cell density in bio-reactor).O 2ratio be applicable to bio-reactor outlet molten O 2level, it is measured by monitor (in Fig. 1 g 6).Via silicone tube or scatterer (Degania Bet, EmekHayarden, Israel), gaseous mixture is supplied to described storehouse.Medium flow is through collecting the separation vessel (in Fig. 1 g 7) of the non-adherent cell in circulation.By peristaltic pump (in Fig. 1 g 9), substratum is circulated.Bio-reactor is equipped with additional sampling spot (in Fig. 1 g 10) and the container for exchanging substratum continuously further.
Produce 3D-adhering substrate cell (3D-ASC)-converge primary people by trypsin treatment by upper described cultivation non-to adhere to 2D cell culture, with the DMEM washing also Eddy diffusion of supplementary 10%FBS, Pen .-Strep-nystatin mixture (100U/ml:100ug/ml:1.25un/ml) and 2mM L-glutaminate, via injection point inoculation (10 3-10 5individual cell/ml) to the 3D carrier of aseptic plug flow reactor (see Fig. 1 g).By PBS-Ca-Mg (Biological Industries before inoculation, Beit Ha ' emek, Israel) bio-reactor is inserted, autoclaving (120 DEG C, 30 minutes), with the Dulbecco growth medium washing containing 10% heat-inactivated foetal calf serum and Pen .-Strep-nystatin mixture (100U/ml:100ug/ml:1.25un/ml).Flow velocity remains on 0.1-5ml/ minute.Seeded process relates to stopping circulation 2-48 hour, makes cell deposition by this on carrier.Under bio-reactor remains on control temperature (37 DEG C) and pH condition (pH=6.7-7.4); Use supply sterile air and CO as required 2incubator.Replace weekly 2-3 secondary growth substratum.Within every 4 hours-7 days, Cyclic culture base is replaced with fresh DMEM substratum.Be 1x10 in density 6-1x10 7time individual cell/ml (after growth 12-40 days), remove total culture volume from bio-reactor, with PBS, bio-reactor and carrier are washed 3-5 time.Then 3D-ASC cell is separated with trypsinase-EDTA from carrier; (Biological Industries, Beit Ha ' emek, Israel; Stir 3-15 minute gently, 1-5 time), after this Eddy diffusion is in DMEM and freezen protective.
3D-ASC quality biomass measures-thaws and counts frozen 3D-ASC cell.For assessment cell survival, by 2x10 5individual cell is inoculated into 150cm 2in tissue culture culturing bottle, assess its adhesive capacity after inoculation in 7 days and heavily form colony (repopulation).After this fluorescent monoclonal antibody flow cytometer (Beckman Coulter, Fullerton, CA) is used to analyze 3D-ASC membrane marker phenotype.
The cytolemma mark spectrum of the adherent cell that 3D and 2D cultivates-by from 100 in 2D culture and 3D flow system culture is compared with Flow Cytometry Assay, 000-200, 000 adherent cell is suspended in the 0.1ml substratum in 5ml pipe, (4 DEG C are hatched with the following various monoclonal antibody (MAb) of saturation concentration, 30 minutes, dark situation): anti-human CD90 (the Chemicon International company that FITC-puts together, Temecula, CA), anti-human CD73 (the Bactlab Diagnostic that PE puts together, Ceasarea, Israel), anti-human CD105 (the eBioscience that PE puts together, San Diego, CA), anti-human CD29 (the eBioscience that FITC puts together, SanDiego, CA), the anti-human CD45 (eBiosience) that Cy7-PE puts together, anti human CD 19 (the IQProducts that PE puts together, Groningen, The Netherlands), the anti-human CD14Mab (IQProducts) that PE puts together, the antihuman CD 34 (IQProducts) that the anti-human CD11b (IQProducts) that FITC puts together and PE puts together or the anti-human HLA-DR Mab (IQProducts) that FITC puts together.After hatching containing 1% heat-inactivated FCS ice-cold PBS in washed cell twice, Eddy diffusion, in 500 μ l0.5% formaldehyde, is analyzed with FC-500 flow cytometer (BeckmanCoulter, Fullerton, CA).
Proteinogram-the ASC being derived from 2D and 3D cultivation program produced from placenta described above of the adherent cell that 3D and 2D cultivates is compared with mass spectroscopy.In brief, by humidification 5%CO 2under environment (atmosphere) in 37 DEG C at 175cm 20.3-0.75x10 is cultivated in culturing bottle 6individual cell 4 days is until reach 60-80% and converge to produce 2D culture.By inoculating 2-10x10 in containing the bio-reactor of 2000 carriers 6individual cell/gram and cultivate over 18 days and produce 3D culture.After results, washed cell (x3) is to remove all serum, and precipitation is also freezing.According to the scheme of manufacturer, [use Tri test kit (Sigma, SaintLouis from throw out isolated protein, USA), with tryptic digestion also with iTRAQ reagent mark (AppliedBiosciences, Foster City, CA)].In brief, iTRAQ reagent is the isobar labelled reagent of non-polymer.To mark the peptide in each sample via its N-terminal and/or lysine side-chain with a kind of in four kinds of isobaric isotope-coded marks.Mix four through the sample of mark, use analytical reagent composition peptide.The quality report ion that often kind of mark release on peptide fragment is unique, therefore, the ratio of four kinds of report ions provides the relative abundance (data is shown in: http://docs.appliedbiosystems.com/pebiodocs/00113379.pdf) of particular peptide in sample.
At Smoler protein group center (department of Biology, Technion, Haifa, Israel) use LC-MS/MS at QTOF-Premier (Waters, San Francisco, CA) upper enforcement is derived from the 2D culture of the ASC of placenta to the Proteomic analysis of 3D culture, and by Pep-Miner software [Beer, I. etc., Proteomics, 4,950-60 (2004)] carry out differentiating and analyzing for the human segmental of nr database.Analyzed protein has: heterogeneity ribonucleoprotein H1 (Hnrph1 in core, GeneBank accession number NP_005511), H2A histone family (H2AF, GeneBank accession number NP_034566.1), eukaryotic cell translation elongation factor 2 (EEEF2, GeneBank accession number NP_031933.1), net calcium binding protein 3, EF-hand shape calcium binding domains (RCN2, GeneBank accession number NP 065701), CD44 isotype 2 precursor (GeneBank accession number NP_001001389, calcium conditioning protein 1 basic smooth muscle (CNN1, GeneBank accession number NP_001290), 3 adenosine phosphate 5 phosphosulfate synthetic enzyme 2 isotype a (Papss2, GeneBank accession number NP_004661), ribosome protein L 7/L a (rpL7a, GeneBank accession number NP_000963) and aldehyde dehydrogenase X (ALDH X, GeneBank accession number P47738).Each experiment carries out 2 times.Because the characteristic analyzed, each protein is analyzed (in analyzing each time, protein occurs 2-20 time) according to the peptide number occurred in the sample to which.
Compare the protein-ASC being derived from 2D and 3D cultural method produced from placenta described above of the adherent cells secrete that 3D and 2D cultivates with ELISA, and 3D cultivates lasting 24 days.After this collection condition substratum, in three independent experiments, with ELISA (R & d system, Minneapolis, MN), Flt-3 part, IL-6, thrombopoietin (TPO) and STEM CELL FACTOR (SCF) are analyzed.Result standard turns to 1x10 6individual cell/ml.
Osteoblast differentiation substratum-assess osteoblast differentiation in 3 weeks by culturing cell in the osteoblast differentiation substratum that forms at the DMEM by supplementary 10%FCS, 100nM dexamethasone, 0.05mM xitix 2-phosphoric acid salt, 10mM B-glycerophosphate.To be dyeed display calcified matrix by Alizzarin Red S, by alkaline phosphatase assay test kit (all reagent from Sigma-Aldrich, St.Lewis, MO) detection of alkaline phosphatase.
Result
PluriX tMbioreactor system creates physiology sample microenvironment
In order to provide effective culture condition to adherent cell, with PluriX bio-reactor (Pluristem, Haifa, Israel; Carrier is illustrated in Fig. 1 g, is shown in Fig. 1 b before inoculation) artificial creation's physiology sample environment (described in Fig. 1 a).As shown in Fig. 1 c-f, the 3D-ASC cell that marrow produces successfully is cultivated and increase after inoculation 20 days (Fig. 1 b-c amplifies x150 and 250 respectively) and 40 days (Fig. 1 c-d amplifies x350 and 500 respectively) in 3D matrix.
The cell of growth in PluriX bioreactor system significantly increase-and the 3D-ASC cell being derived from placenta of different production batch grows in PluriX bioreactor system.Inoculum density is 13,300 cell/carriers (is 2x10 to total amount 6individual cell).Inoculate after 14 days, cell density is increased to 15 times, reaches about 200,000 cell/carrier (Fig. 2), in other words 30x10 in the bio-reactor of 150 carriers 6.In different experiments, by cell with 1.5x10 4cell/ml density is inoculated into bio-reactor, and inoculate after 30 days, carrier contains the larger cell quantity more than 50 times, i.e. about 0.5x10 6individual cell/carrier or 0.5x10 7individual cell/ml.Cell density on the carrier of various horizontal growth post is consistent, oxygen is described and nutrient substance is homogeneous is delivered to cell.Prove that 3D culture systems is that the growth of high-density mesenchymal cell culture and long term maintenance provide support condition thus, described cell culture can effectively grow into be enough to be used in supporting moving into and the amount of successful implantation object.
The unique membrane marker feature of 3D-ASC display-in order to determine that shla molecule secretion profile and protein produce the difference of (being implemented by the 3D cultural method of analog bone environment), carry out FAC analysis.As shown in Figure 3 a, the facs analysis of cell marking describes, and 3D-ASC shows the marker expression pattern different from the adherent cell grown in 2D condition.Compared with 3D cultured cells, 2D cultured cells expresses obvious higher levels of positive membrane marker CD90, CD105, CD73 and CD29 membrane marker.Such as, the CD105 of 3D cultured cells display 56% expresses, and contrast 2D cultured cells is 87%.The ASC of both 2D and 3D placenta culture does not express any hematopoiesis membrane marker (Fig. 3 b).
Soluble factor spectrum-hematopoiesis the tabernacle of 3D-ASC display uniqueness comprises the sustenticular cell producing and enrich cytokine, chemokine and somatomedin.In order to determine the difference between the ASC that 2D and 3D cultivates further, to obtain in 2D and 3D ASC culture conditioned medium four kinds of main hematopoiesis secretory protein spectrums by ELISA.Fig. 4 a-c show growth the cell of 3D condition produce containing higher levels of Flt-3 part (Fig. 4 a), the conditioned medium of IL-60 (Fig. 4 b) and SCF (Fig. 4 c), and detect in the conditioned medium of 2D culture lower level IL-6 and close to the Flt-3 part of zero level and SCF.In two kinds of cultures, thrombopoietin (TPO) output is all very low and equally matched.
3D-ASC shows unique proteinogram-in order to determine the difference between the ASC that 2D and 3D cultivates further, with the proteinogram of these cells of mass spectroscopy in mass spectroscopy.Fig. 4 d represents that the ASC that 2D and 3D cultivates shows visibly different protein expression profile.As shown in table 1 below, 3D cultured cells shows higher H2AF and ALDH X expression level (exceeding respectively more than 9 and 12 times) and higher EEEF2, RCN2 and CNN1 protein level (being respectively about 3,2.5 and 2 times).In addition, 3D cultured cells represents Papss2 and the rpL7a expression level of only about half of albumen Hnrph1 and CD44 isotype 2 precursor expression level and about 1/3rd.
Table 1
3D-ASC has the osteoblastic ability-in order to represent the feature of 3D-ASC further of being divided into, by cell cultures 3 weeks in osteoblast differentiation substratum.After this calcium deposit is implemented.The cell display of differentiation creates calcium (redness in Fig. 5 a-b is described), otherwise compared with control cells is maintained in fibrocyte sample phenotype and shows not have mineralising (Fig. 5 c-d).It is osteoblastic ability that the 3D-ASC that these results show to be derived from placenta has vitro differentiation.
Embodiment 2
The 3D-ASC being derived from placenta improves the capability evaluation of HSC immigration
By detecting human hematopoietic cell (hCD45+) level in sub-Lethal irradiation or the pretreated immune deficiency NOD-SCID mouse of chemotherapy, assessing 3D-ASC and supporting that HSC moves into.
Material and experimental technique
Separation of C D34+ cell-get cord blood sample (BneiZion Medical Center at farrowing interval under aseptic condition, Haifa, Israel), with Lymphoprep (Axis-Shield PoC As, Oslo, Norway) density gradient centrifugation layering monocyte freezen protective.Wash the monocyte thawed and also use AntiCD3 McAb 4 antibody incubation, be separated with midi MACS (Miltenyl Biotech, Bergish Gladbach, Germany).Cell from more than one sample is merged and reaches required amount (50,000-100,000 cell).
To detect 7 weeks age of the cell of transplanting-support in aseptic open system cage in radiation murine male with female NOD-SCID mouse (NOD-CB 17-Prkdcscid/J; Harlan/Weizmann Inst., Rehovot Israel), give aseptic diet and autoclaved sour water.Sub-lethal (350cGy) radiation murine, after this (after radiation 48 hours) are by intravenous injection to tail vein, transplant 50,000-100,000 hCD34 +cell, adds or does not add the other ASC (0.5x10 being derived from placenta or fatty tissue 6-1x10 6) (often organizing 3-7 mouse).After transplanting 4-6 week, mouse is put to death in dislocation, rinses collection BM with FACS damping fluid (50ml PBS, 5ml FBS, 0.5ml sodium azide 5%) from Thigh bone and shin bone two.By the people's cell in Flow cytometry mouse BM, by making cell and anti-human CD45-FITC (IQ Products, Groningen, The Netherlands) hatch, produce the cell percentages of expressing people and mouse CD45 hematopoietic cell markers in the NOD-SCID mouse of process.Clear and definite people moves into minimum valve limit and is appointed as 0.5%.
The detection-as the above-mentioned male NOD-SCID mouse (NOD.CB17/JhkiHsd-scid in 6.5 week age for supporting radiation murine of transplanted cells in the mouse of chemotherapy process; Harlan, Rehovot Israel) accept busulfan (continuous 2 days of 25mg/kg-) peritoneal injection.Second time injection busulfan after 2 days, alone CD34+ cell or with the 0.5x10 produced from placenta 6individual ASC injects mouse together.After transplanting 3.5 weeks, as there is situation to what measure human hematopoietic cell as described in radiation murine above in execution mouse.
Result
The HSC that 3D-ASC improves radiation murine moves into-at co-transplantation people CD34+ hematopoietic cell in radiation NOD-SCID mouse and the 3D-ASC being derived from placenta or fatty tissue.After co-transplantation, assessment in 4 weeks moves into efficiency, and compares with the mouse that alone HSC transplants.As shown in table 2 and Fig. 6, compare with the mouse of alone UCB CD34+ cell process, 3D-ASC and UCB CD34+ cell co-transplantation causes the level height of people's cell in the high quite a lot of and recipient mice BM of immigration rate quite a lot of.
Table 2
The cell transplanted Average h-CD45 STDEV
CD34 3.8 7.9
CD34+ is from the 3D-ASC of placenta 5.1 12.2
CD34+ is from the 3D-ASC of fatty tissue 8.7 9.6
The HSC that 3D-ASC improves chemotherapy mouse moves into-by people CD34+ hematopoietic cell be derived from 500,000-2D-ASC or 3D-ASC co-transplantation of placenta in the pretreated NOD-SCID mouse of chemotherapy.
After co-transplantation, assessment in 3.5 weeks moves into efficiency, and compares with the mouse that alone HSC transplants.As shown in table 3, compared with alone UCB CD34+ cell, ASC and UCB CD34+ cell co-transplantation causes the immigration higher level in recipient mice BM.In addition, as shown in table 3, be used in the mouse of adherent cell (3D-ASC) co-transplantation being derived from placenta grown in PluriX bioreactor system, than the average immigration higher level of the mouse of the cell co-transplantation from same donor of growth in common static 2D culture condition (culturing bottle).
Table 3
The cell transplanted Average h-CD45 STDEV
CD34 0.9 1.1
CD34+ is from the conventional 2D culture of placenta 3.5 0.2
CD34+ is from the 3D-ASC of placenta 6.0 7.9
Facs analysis result shown in Fig. 7 a-b proves the advantage of ASC and hHSC (Fig. 7 b) co-transplantation, and ASC improves the ability of hemopoietic system recovery after HSC transplants.
These results integrate and show, ASC improves Radiation in jury as sustenticular cell can transplant (autologous or allogeneic) at HSC after.3D-ASC improves ability that hemopoietic stem cell and/or progenitor cell move into can improve the going back to the nest of transplanted cells, self and the multiplication capacity ability of cytokine of support HSC from 3D-ASC secretion after HSC transplants, or goes back to the nest from the transplantable HSC of those cellular reconstitutions and breed the ability of required impaired hematopoieticmicroenviron-ment.
Embodiment 3
The ASC cultivated by 2D and 3D suppresses lymphocyte responses
Find in MLR analyzes adhering substrate cell especially 3D-ASC suppress the immune response of Human Umbilical Cord blood monocyte.
Material and experimental technique
Mixed lymphocyte reacion (MLR) measures-is measured by MLR assay method immunosuppression and immune privilege (immunoprivilege) characteristic of the ASC from placenta generation being derived from 2D and 3D cultural method, MLR measures to measure and is positioned at the histocompatibility of HLA locus, realizes according to incompatible lymphocytic proliferation rate in the mixed culture promising cell (hyperplasia) and irritation cell (can not hyperplasia).End user's Cord blood (CB) monocyte (2x10 5) as responsive cell, its by with equivalent (10 5) co-cultivation that combines of the adherent cell produced by placenta being derived from human peripheral blood mononuclear cell (PBMC) or cultivating with 2D or 3D crossed of radiation (3000Rad) or adherent cell and PBMC stimulates.Each measures in triplicate.The RPMI1640 substratum of cell in 96-orifice plate is (containing the 5%CO of 20%FBS at humidification 2environment is in 37 DEG C) in co-cultivation 4 days.At last 18 hours that cultivate with 1 μ C 3this plate of H-thymidine pulse.Then through glass fibre filter collecting cell, absorb with the quantitative thymidine of scintillometer.
Result
Fig. 8 shows the immunne response of CB cell, and this shows according to the propagation increase of these cells when stimulating with PBMC, and under not being bound by any theory, this probably breeds relevant with the T cell of the uncompatibility of response HLA.But when hatching with adherent cell of the present invention, these cells demonstrate quite low immunne response level.In addition, when jointly hatching with these adherent cells, the immunne response of CB to PBMC substantially reduces.Therefore, in the mode similar to MSC, find that ASC has the ability of T cell propagation (typically GvHD) of potential reduction donor's cells.Although 2D and 3D two kinds of cultures all reduce lymphocyte immunity response, consistent with other advantage of 3D-ASC mentioned above, the immunosuppression of 3D ASC is stronger.
Should understand, for clarity sake, some feature of the present invention set forth in the context of embodiment out of the ordinary also can combine and provide in single embodiment.Otherwise for the purpose of brief, the of the present invention various feature set forth in single embodiment context, also can separate or provide with any suitable sub-combination.
Be elaborated although the present invention combines its specific embodiments, obviously much alternative, modifications and changes to those skilled in the art will be apparent.Therefore, all alternative, the modifications and variations like this in the spirit dropping on accessory claim and broad range are intended to comprise.All publications, patent and patent application and GenBank registration number that this specification sheets is mentioned draw at this reference making this specification sheets with its entirety, and its degree is as clear and definite and pointing out each independent publication, patent or patent application or registration number to be hereby incorporated by reference individually.In addition, adding, quote or admitting of any reference in the application should not be understood to admit that such reference can be used as prior art of the present invention.

Claims (8)

1., from the adhering substrate cell mass amplification method of placenta or fatty tissue, described method comprises:
Cultivate adhering substrate cell mass under i three-dimensional cultivation condition that () is increased at sustenticular cell, wherein, described three-dimensional cultivation condition comprises the growth matrix with nanoscale cell epimatrix fiber, so that the foundation structure of simulated tissue; With
(ii) from three dimensional culture system, increased adhering substrate cell mass is collected,
Wherein, described adhering substrate cell mass is expressed CD73, CD90, CD29 and CD105 but is not expressed CD45, CD80, HLA-DR, CD11b, CD14, CD19, CD34 and CD79.
2. the method for claim 1, it comprises the substratum collecting the adhering substrate cell mass cultivated through amplification further, and preparation comprises Flt-3 part, the conditioned medium of IL-6 and SCF thus.
3. the adhering substrate cell mass through amplification of the method generation of claim 1.
4. the adhering substrate cell mass through amplification of claim 3, wherein, described adhering substrate cell mass Immunosuppression reaction.
5. the conditioned medium prepared of the method for claim 2.
6. pharmaceutical composition, it comprises the adhering substrate cell mass through amplification and pharmaceutically acceptable carrier of claim 3.
7. the process of claim 1 wherein that described three dimensional culture system comprises 3D bio-reactor.
8. the process of claim 1 wherein, cultivate described adhering substrate cell mass and reach at least 60% and converge.
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