CN1763173A - Hematopoietic stem cell culturing apparatus and method - Google Patents
Hematopoietic stem cell culturing apparatus and method Download PDFInfo
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- CN1763173A CN1763173A CN 200510028019 CN200510028019A CN1763173A CN 1763173 A CN1763173 A CN 1763173A CN 200510028019 CN200510028019 CN 200510028019 CN 200510028019 A CN200510028019 A CN 200510028019A CN 1763173 A CN1763173 A CN 1763173A
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Abstract
The present invention relates to apparatus and method of culturing mammal cell, and is especially culturing apparatus and method for selective amplification of human stem cell, including hemopoietic stem cell. The apparatus and method of the present invention may be used widely in biological treatment of tumor and transplantation of hemopoietic stem cell.
Description
Technical field
The present invention relates to the apparatus and method that a kind of mammal cell is grown in cultivation, relate in particular to the culture apparatus and the method for human stem cell of a kind of selectivity amplification in vitro and hemopoietic stem cell.These apparatus and method can be widely used in tumor biotherapy and hematopoietic stem cell transplantation.Can be widely used in all kinds of clinical biological cell therapeuticals.
Technical background
Cell is widely used for different therapeutic purpose, and its possibility is the emphasis that biomedical boundary pays close attention to all the time.The human body hemopoietic system produces various kinds of cell and avoids pathogenic agent, toxicant and other deleterious infringements with the protection human body.Hemopoietic system originates from simple stem cell, induce the generation hemopoietic system by stem cell, principle and process that how human stem cell is induced to differentiate into hemopoietic system are not understood fully as yet fully, in case and stem cell forms hemopoietic system, can produce some factors, for example transplant stimulating factor etc., these factors may promote that development of stem cells is sophisticated hemocyte.
Hemocyte comprises red corpuscle, white corpuscle and thrombocyte three class cells, and they all originate from hemopoietic stem cell.Hemocyte has a lot of purposes, and thrombocyte is also producing PDGF to the antihemorrhagic while.The red corpuscle carrying is shifted oxygen molecule to each tissue, and different lymphocytes can be used for treating multiple disease, resists certain responsive especially pathogenic agent specifically.Stem cell has been used to gene therapy, tumour high-dose chemotherapy at present or/and the supportive treatment of radiotherapy also can be used for the tumor vaccine treatment for the basis of immunotherapy of tumors and dendritic cell.
As everyone knows, hemopoietic stem cell is grown in bone, is broken up in various degree, wherein partly is to take place in marrow.Thereby recreate a system, and provide similar marrow same environment, in this environment, can cultivate with induced growth and go out specific cell family, such system all can be highly significant to research and clinical application.
Organizational engineering is that emerging and development is rapidly partly in the biotechnology, its target is to build all or part bodily fuctions sex organization at external environment again, make it to satisfy various clinical application or other needs, a lot of research is just in evolution, its target in external environment partly or all duplicator's body weight to organize, perhaps artificial skin belongs to the most successful example in the initiative work.
For a long time, the system at external mass production human bone marrow and other hemopoietic stem cells is developed in the expectation of bio-science man, because such system can not only satisfy extensive and diversified clinical needs, simultaneously also can provide great help for scientific research and applied research, its application is as follows:
The elementary dynamics research of 1 hemopoietic stem cell differentiation
2 improve from body or simplified marrow transplanting
3 emptyings or remove any cell of occurring of not wishing in bone marrow transplantation are as T cell or other fatal cells
The gene therapy of 4 blood cell systems
5 improve miscellaneous tumour cell in human body autologous peripheral blood stem cell transplantation, the purification stem cell
6 improve navel blood stem cell transplanting, amplification navel blood stem cell quantity, to satisfy the needs of adult's oncotherapy
7 are used to induce the differentiation dendritic cell, carry out immunotherapy of tumors and the tumor vaccine treatment of dendritic cell for the basis
8 a large amount of human hematopoietic stems that produce are as CD34
+Cell uses for clinical or research
All kinds of mature blood cells of 9 a large amount of generations are as red corpuscle, thrombocyte.
The later stage eighties has proposed long-term hemopoietic stem cell and has cultivated (" Long-term Hematopoietic culture " " LTHBMC "), and recent development has significantly improved original method, and has produced revolutionary change.
Yet these improvement remain the subclinical quantity medullary cell implemented or the method for other hemopoietic stem cells under the tissue culturing equipment of standard laboratory scale.
In the vitro culture environment, stroma cell is supported the ability of human hematopoietic stem to be proved to be can be cultivated exchange rate by media apace to improve, in exchange of fast culture media and highdensity cell, LTHBMC can support a large amount of stable immature cells that produce in the hemopoietic stem cell to reach for 20 weeks, these evidences prove, under above-mentioned culture environment, can stably produce hemopoietic stem cell in a large number.
Correctly adopt and further to improve these cultured method by the dissolved cell growth factor.
In order to overcome the deficiencies in the prior art, must develop the composing method and the device that can reach clinical quantity.
In the bio-reactor design in the past, although being used as important aspect, the cell harvesting program considers, but the method that proposes remains the structure of setting up in a wide range shape of the mouth as one speaks exploitation formula, when harvested cell, must stop culturing process, and must expose the cultivation chamber in atmosphere, this structure is easy to constitute the pollution of cultivating chamber.
In cell cultures, bio-reactor need keep a kind of equilibrium state of inside, especially in the initiating cell culturing process of complexity, human stem cell or mankind hemopoietic stem cell are to nutrition speciality filling rate very responsive (being gas filling rate/nutritive medium filling rate/somatomedin filling rate), also very responsive to the supply and the removal of above-mentioned substance simultaneously, responsive equally to the chemical environment of cultivating, in the present bio-reactor, can satisfy above-mentioned requirements without any design, thereby keep cultivating homeostasis and stable, especially in the initiating cell culturing process.
Also fail to propose a kind of method harvested cell under the state that does not interrupt cultivating in the present design, equally do not have a kind of design can keep cultivating homeostasis yet, possess the gas that provides stable and the method for these two kinds of advantages of liquid filling rate simultaneously and more do not find.
The purpose of this invention is to provide a kind of totally enclosed, HT rate, high media cultivation exchange rate, the hematopoietic stem cell culturing apparatus of harvested cell under the state that does not interrupt cultivating.
Summary of the invention
In order to realize purpose of the present invention, the technical solution adopted for the present invention to solve the technical problems is:
A kind of design of bio-reactor is provided, can provide the preceding cell of human stem cell or stem cell to comprise cultivation, the growth of complicated preceding initiating cell, keep homeostasis, keep stable gas liquid to pour into and remove, can when harvested cell, not interrupt culturing process again.
This device can continue to provide perfusion, and can produce and obtain high-density and great-hearted hemopoietic stem cell.
This method and apparatus can induce, increase at external environment human stem cell and hemopoietic stem cell family, this effective amplification is particularly useful for hemopoietic stem cell and early stage progenitor cell, this method can be used for specific clinical application, and can continue, regular or interim harvested cell.
Technical characterictic of the present invention is, a kind of bio-reactor of cultivating human stem cell and human hematopoietic stem that is exclusively used in is provided, forms by a sealing, cultivation chamber that boundary is clear and definite, in this chamber, human stem cell or hemopoietic stem cell can be induced and be cultivated, and are made up of following part.
● the semi-permeable membranes of a gas-permeable, liquid-impermeable body suitably is placed on this chamber, thereby this chamber is divided into a gas chamber, a cultivation chamber.In gas chamber, exchanging gas enters the cultivation chamber of opposite side by semi-permeable membranes.
● the liquid cell culture fluid enters the cultivation chamber by the radial structure guiding inboard at inlet position, the nutrient solution that was used is then by the outer side system of the guiding of outlet position radial structure, therefore cultivating the perfusion passage that forms without hindrance, a radial nutrient solution between chamber import and the outlet.
● partial structure is by the gas of above-mentioned gas chamber's perfusion cellular respiration.
● partial structure is used for continuing, regularly or interimly gather in the crops non-adherent cell from cultivate chamber.
Technical characterictic of the present invention is, bio-reactor provided by the invention is suitable for cultivates human stem cell or hemopoietic stem cell, and is waterproof but can be through the film of gas at the semi-permeable membranes of this employing.
The bio-reactor of this suitable cultivation human stem cell or hemopoietic stem cell, its cell cultures chamber, the part that comprises suitable cell adhesion in a surface and growth is used for another continuing, the regular or interim part of gathering in the crops non-adherent cell, back some is positioned at outlet, its guiding liquids cell cultures nutritive medium moves in the culture chamber indoor circulation.
The structure of above-mentioned results non-adherent cell optionally harvested cell is based on the density of cell.
The culture chamber chamber surface has a panel region cell adhesion.
The above-mentioned suitable cell adhesion or the surf zone of attaching are that bioactivity surface is exclusively used in the specific cells adhesion.
The above-mentioned suitable cell adhesion and the film of growth are suitable for the cell cultures chamber and place gas semi-permeable membranes top.
Cell culture chamber is indoor, constituting an obstruction liquid culture media mobile between the film of gas semi-permeable membranes and above-mentioned suitable cell adhesion/growth separates, the cell cultures chamber is the offside that is positioned at cell adhesion, and gas chamber is positioned at the offside that obstruction liquid culture media flows and separates.
Technical characterictic of the present invention is that human stem cell and human hematopoietic stem amplification system are made of following:
● the gas semi-permeable membranes in above-mentioned chamber, is divided into gas chamber and cultivates chamber.Exchanging gas in air chamber sees through the gas semi-permeable membranes and enters the cultivation chamber.
● radial entrance structure and radial export structure form the passage of a radial liquid-flow.
● dabbling cellular respiration gas enters the cultivation chamber by air chamber.
● a part of structure can be gathered in the crops non-adherent cell, is made up of following part:
1. a storage facility structure can retaining liquid shape cell culture media.
2. first pump structure can enter the cell cultures chamber by the perfusion fluid cell culture media.
3. the source of cellular respiration gas.
4. second pump structure can pour into cellular respiration gas by the cell cultures chamber.
Technical characterictic of the present invention is,
The source of cellular respiration gas is a kind of compressed mixed gas.
The source of cellular respiration gas is a kind of cultivation incubator.
Perfusion enters and comprises a kind of moisture in the cellular respiration gas, and air is by humidification before being fed into the cultivation chamber.
First kind of pump structure is the liquid filling pump, and the fresh liquid cell culture media of this perfusion enters cell culture chamber, and extracts used aqueous cell media out from the cell cultures chamber.
Cell adhesion/growth film is suitable for the cell cultures chamber, and is positioned at ventilative semi-permeable membranes top.
Gas semi-permeable membranes and cell adhesion film are separated gas chamber and are cultivated chamber, and liquid media separate the offside that is positioned at cell adhesion/growth film.
The cell cultures chamber comprises a kind of film of energy adhesion/one-tenth growing cells.
The method that selectivity results non-adherent cell is selected based on cell density.
Hinder the mobile offside that is positioned at cell adhesion/growth film that separates of liquid culture media, with offside with air chamber.
The gas semi-permeable membranes can see through gas but can not see through liquid.
The used antibody of bio-reactor is to help out for immature cell.
Above-mentioned antibody is CD34
+Antibody.
Above-mentioned antibody is distributed in around the gas semi-permeable membranes.
The invention has the beneficial effects as follows provide a kind of full automatic control, sealed, automatically change nutrient solution, gather in the crops the device of culturing cell automatically, for external hematopoietic stem cell expansion provides a fabulous instrument.
Description of drawings
Accompanying drawing 1 is a perfusion chamber synoptic diagram
Accompanying drawing 2 is ring junction compositions among Fig. 1
Accompanying drawing 3a, 3b are radial fluid chamber structure iron
Accompanying drawing 4a, 4b are the growth chamber synoptic diagram
Accompanying drawing 5a, 5b are the bio-reactor shape assumption diagrams
Accompanying drawing 6 is flat, bio-reactor synoptic diagram
Accompanying drawing 7 is Fig. 6 Zhong Yuan tapering part synoptic diagram
Accompanying drawing 8 is apsacline bio-reactor synoptic diagram
Accompanying drawing 9 is bio-reactor amplification system schemas
Embodiment
The invention will be further described below in conjunction with drawings and Examples.
Fig. 1 is a perfusion chamber synoptic diagram.Reactor 10 has top cover dish shape thing 12 and bottom cover dish shape thing 14.By screw 16 combinations, wingnut 18 fixed positions, these three screws are used avoids flexural deformation, chamber 20 has three parts, the substrate that pars intermedia 22 has support is the basal cell of basal cell and implantation, and medullary cell, central part 22 is divided into top portion 24 and bottom part 26, tunicle or reticulattion 28 and 30 separate respectively, the film that many carbon compounds constitute may be used, perhaps a kind of stainless reticulated structure filter screen, this hole is very tiny, (adopts laser boring technology, makes and can breathe freely but liquid-tight film) so that cell can be contained in the intermediate cavity, the internal procedure of separating adopts inner garden post 27 to be placed in the chamber, is used as the mechanical support effect of the demarcation membrane of part between compartment.Top portion 24 does not need identical structure with bottom part 26, and pipeline or film intersection will be arranged, and wherein liquid culture media and gas will be exchanged at that time.
The silicone tube of gaseous interchange by intersecting, the length of pipeline can be changed to allow enough gas flows to cultivate with sustenticular cell, and these cells are in the intermediate cavity metabolism.
Cultivate media can be poured or be sucked directly from the top or the bottom by 32 partly, or may be fed by carrying pipe 34.
If desired, top and bottom partly can be limited, by using a kind of outside apparatus of oxygen supply, at this state, separatory membrane is fixed on post top, glass garden, this glass garden post matches with garden indented in columnar dish 12 and 12, is used to allow the high-quality fluid to pass across membrane structure at garden indented in columnar interior region.
This geometrical layout allows fluid from the inlet maximum quantity that limits to a number or amount partly, and for the radial pressure of equilibrium, another uniform amount of liquid passes through demarcation membrane, this set is suitable for having contained in the chamber small amounts of cells of relative populations, so that oxygenation can not be restricted.
Fig. 2 is a kind of ring texture that statement forms that illustrates, and it connects the cultivation media chamber of filling cavity to the side, aeration device, and sensor cavities, and sample/injection is partly.
An exogenous fresh culture media 50 is filled into by pump 52 by pipeline 56 cultivates the media storage pool, is filled into by pump 52 from storage pool 54 and uses cultivation media storage pool 60 to be for further processing.
Second pump 62 suction media passes through a tubular fibre aeration device 66 from media storage pool 54 by pipeline 64, cultivates media and directly entered first chamber 70 of bio-reactor by pipeline 68.
Suitable cultivation composition 82 is provided by injection system, is transferred to first chamber 70 of bio-reactor by pipeline 68 with cultivating media, and cultivating composition 82 may be test composition or the interpolation factor or other similar compositions.
Cultivate media and directly enter second chamber 74 by central bore 72 from first chamber 70 of bio-reactor, same, cultivate media and can arrive pipeline inner sensor 78, to survey the variation of cultivating composition in the media component by pipeline 76.
For example, L-GLUTAMICACID amine: glucose ratio is 1: the 5-8 scope can be deemed appropriate, depends on the use of cell line, to the 3T3 cell, 1: 8 ratio is optimum transfer, further, it is optimum that ammonia accumulates in 2mm, assembles in the part and must be less than 35mm.
By monitoring effusive liquid in the bio-reactor, thereby regulate the cultivation media of input bio-reactor, oxygen partial pressure also may be changed, the gas flow ratio also may be conditioned, various compositions may be increased, or the perfusion ratio is become slowly perhaps increase.
From transmitter 78, cultivate media and directly be filled into liquid storing pool 54 by pipeline 80 by pump 62.Pass through by above-mentioned liquid-flow, cultivation media at a side liquid storing pool is slowly exchanged, by a pump that separates, this organizational structure allows separately control to cultivate the exchange rate (periphery pump) and the flow velocity ratio (by oxygen exchange device and perfusion chamber) of media, the former is used for control cultivation media formation and dabbling long-term change, and the latter can change the flow rate of liquid form of the tension force and the chamber of oxygen.
Adopt the biocompatible membrane of fine mesh to allow the aperture fluid to enter chamber, thereby allow clear and definite cell growth factor and other special compositions to be respectively applied for hemopoietic stem cell or basal cell.
Behind other components of the whole cultivation chamber of disinfection with high pressure steam and this ring texture, bio-reactor is supposed a gnotobasis, and the monitored while of every signal in the training period, cultivating media may circulate a couple of days by ring texture and chamber.
Suppose that sterilizing process is done, the central part of chamber or be poured by extra cytomixis composition or the composition that contained stroma cell.
These cells are gathered in the crops by syringe pump, perhaps allow cell to continue to flow out chamber, owing to cultivate the injection pressure of media, flow out by output channel.
Fig. 3 a, radial fluid cavity 100 among the 3b, inlet 102 and outlet 104 are arranged, the direction that cavity 106 is pressed arrow 108 sensings flows, hemopoietic stem cell 110 is inoculated into stroma cell layer 112, grows in cavity, and flow velocity will be detected, can be adhered to until cell, non-adherent cell 114 flows out by exporting 102.
Fig. 4 a, growth chamber 120 has inlet 122 and outlet 124 among the 4b, and inlet 122 is made up of multiple manifold 128, and multiple manifold 128 is supplied indivedual culture chambers 126, contain hemopoietic stem cell 110 and stroma cell 112 in single culture chamber 126, cell is grown in growth chamber 126 and is separated.
Fig. 5 a cultivates in the chamber among the 5b and blocks bar 134,136, and 138 are progressively removed in cultivation, block bar 134 in 8～10 weeks, and 136 in 18～20 weeks, and 138 in 28～30 weeks.
Bio-reactor be described to four multi-form:
I. flat bed (hemopoietic stem cell) bio-reactor (figure .6a-6e)
Ii. the harvested cell sample of a taper of flat bed (hemopoietic stem cell) bio-reactor band partly (Fig. 7)
Iii. (hemopoietic stem cell) bio-reactor (Fig. 8 a, 8b) tilts
Iv. one on the longitudinal axis formula inclination bio-reactor band fluidic device that is tapered
The single or multiple surfaces of flat bed (hemopoietic stem cell) bio-reactor band.This bio-reactor is at least by two machineries or the flat accessory of die casting, 600 and 601 usefulness nontoxicitys, the material manufacturing that can allow cell grow up, for example: poly-stupid ethene (PolyStyrene, Polycarbonate, Polysufone etc.), form the bio-reactor top 600 and the end 601, on the top 600 and the end 601 two packing rings 602 are arranged, this can be with any nontoxicity, the material that can grow for cell silica gel for example.
When bio-reactor is assembled, a film 603 is placed between two packing rings 602, separately push up 600 and the end 601 during combination, whole combination can adopt any known mode to be embedded into by screw with clip or with screw hole 606, yet the configuration of other screws also is possible.
After assembling, the structure of two chambers of a dead front type band forms, one as cell cultures 614, another is a gas chamber 615, and bio-reactor top 600 has different parts with the end 601, at Fig. 6 a-6e, 601 show two gases parts 607 and 608 at the bottom of the bio-reactor, one is the gas input, and another is discharged for gas, shows three parts at bio-reactor top 600.A liquid media flows into part 610, liquid flows out partly 611 and cell samplings or results, and partly 609 (this partly may be with a piston, in case stopping leak leaks), output partly 611 is configured no zero angle formation, main surface plane source for top 600, help any non-adherent cell and under the gravity guiding, flow out cultivation chamber 614 smoothly, thereby enter outlet 126, be labeled in above the FIG6, the geometricdrawing hole that is positioned on the packing ring 613 is a kind of annular, but in a kind of flat ellipse garden shape or other configured openings, be positioned at the entrance and exit joint area, this shape has and helps provide the output of better liquid, and similarly geometricdrawing can be used to obtain ideal scissors pressure distribution and help flowing of liquid.
In addition, similar configuration only has a packing ring 602 not have film 603 to use.In this state, annular seal space can not be separated into lumen and air chamber, cultivates the media perfusion then by position, input aperture 610, imports indispensable cellular respiration gas simultaneously, and the flat bed bio-reactor can be assembled into four basic configurations.
Configuration 1: Fig. 6 f
Two intervals are provided, cell cultures chamber 614 and gas chamber 615, a double-side membrane assembling separates two chambers, and (for example a kind of porcelain shape material membrane is used for cell growth/adhesion 605 and puts waterproof and breathables, can be above the film 604 of air inlet body exchange, pellosil for example), at cell cultures chamber 614, the liquid culture media is poured in the exchange position at liquid culture media inlet position 610 and outlet position 611, meanwhile, gas is by air chamber 615, in position, gas inlet 607 and the position 608 cross-linking part circulation of gas export department.Cell 612 is grown in porcelain shape material cell growth/adhesion 605 surfaces of cultivating chamber 614.
Configuration 2: Fig. 6 g
This is a kind of and above-mentioned pattern of putting upside down.Cell is grown in the surf zone 612 of bio-reactor bottom 601, in the bottom chamber 614 of culture chamber, extremely be fit to cell adhesion/growth surface zone 625, in this configuration, an independent gas-exchange membrane 604 is only arranged, do not need to adhere to and the film 605 that becomes growing cells.The liquid culture media is poured by cell cultures chamber 614 and gas perfusion and passes through gas chamber 614.
Configuration 3: Fig. 6 h
The design of a kind of three chambeies, gas is by chamber 615 circulations, gas chamber 615 is positioned at the bio-reactor top, separate by gas-exchange membrane 604 and cell growth chamber 614, cell 612 is grown up in cell growth chamber, and growth chamber 614 is by cell adhesion/growth, and film 605 separates every 616 with liquid culture media chamber, cell cultures chamber 614 is hindered in this configuration, when the liquid culture media hinders 616 through the input aperture partly 610 and delivery port partly 611 when being continued perfusion.
Configuration 4: Fig. 6 i
The design of three chambeies, in this framework, be the modification of configuration 1, by place one the 3rd packing ring 602 between gas-exchange membrane 602 and cell adhesion/growth film 605, this collocation form has increased by one and has hindered liquid media mobile chamber 617 between air chamber 615 and cell bed 612.
In order to prevent the migration of gas agglomerate, the leakage of attaching and anti-sealing at the bottom of cell/extra cell based, a kind of two-layer film configuration, being indicated in Fig. 6 f, is that ideal is used, lower membrane 604, it is waterproof, prevent that moisture content from leaking and permission gas passes through, film 604 provides mechanical support with respect to upper layer film 605, and upper layer film 605 is for cell adhesion and growth, may be that a kind of inorganics ceramic material constitutes, it is white that it may be coated with the extra cell dawn of one deck.Peptite-2000RGD TeleosPharmacenticals San Diego Poly for example.Further, the non-organic film of a kind of high-performance is used, thus it can penetrate hydrated compound make microscopic examination and the monitoring cell be grown to serve as possibility.
Cultivate media and gas to bio-reactor for providing, biological respinse top 600 and bottom 601 connected pipelines can adopt any known suitable material, as Propylene silica gel etc., and the joint that can provide and do not produce leakage.
The schema of a bio-reactor amplification system shown in the accompanying drawing 9 is included in other needed accessory in service.
Liquid media 900, remain on freezing state (probably about 4 ℃), to prevent the unsettled component corruption of Chemical Composition, as L-GLUTAMICACID amine (and somatomedin) by the cell cultures chamber 614 of pump 901 (can be syringe pump, peristaltic pump etc.) pump to bio-reactor 902, by pipeline 903, this requirements for pipes waterproof is to prevent that cultivating medium changes before entering cell cultures chamber 614.Pipeline 903 device that can slow down like this, can move between pump 901 and/or the fresh liquid media storage pool 900.Any treating processes is all in gnotobasis between refrigerated storage space 910 and sheet metal fluid lid (not shown).Other pipeline 903 is maintained at (not shown) in the refrigerator, and like this, only a bit of pipeline is exposed in room temperature or the incubator temperature.
Gas is supplied to the gas chamber 615 of biological respinse 902, (1～50% mixes perhaps to contain pre-mixed gas from taper 904, be preferably in 5～20% (V/V) O.Sub25% (V/V) COSub2, or simply from (not shown) in the incubator (typical mixing air and 5% (V/V) CO.Sub2).The flow velocity of gas and composition are easy to control, gas can adopt a pump to cultivate warming apparatus 906 by one by standard cell lines and heat before the fortune body transports gas chamber 615 to, 100% temperature is possible, and gas pipeline 907 can selectivity contain sterilizing filter 905.Used cultivation media can be by collection device 914 in piping 909 be collected in receiving flask 908.The sample of used nutrient solution can be extracted out in receiving flask 908, and to analyze the composition of cultivating media, used gas is discharged from by pipeline 911, and expellant gas also can be analyzed with the gas analysis apparatus of any sky.
Analyze used cultivation media and used gas, may be very helpful, adopt device 912, monitor PH or oxygen partial pressure, might help to determine cultural characters the monitoring cell cultivation process.
The gas semi-permeable membranes can adopt (Silicone membrane, specification, perhaps a kind of Teflon, RTMmemebrane (of 0.001 inch thickness) such as pellosil.Cell adhesion/growth film employing porcelain shape material membrane (ceramic membrane, Anotec, RTM, 0.02micron, nontreated).
Claims (5)
1. the invention provides a kind of design of bio-reactor, can provide the preceding cell of human stem cell or stem cell to comprise the cultivation of complicated preceding initiating cell, grow up, keep homeostasis, keep stable gas liquid to pour into and remove, can when harvested cell, not interrupt culturing process again, this device can continue to provide perfusion, and can produce and obtain high-density and great-hearted hemopoietic stem cell, this method and apparatus can be induced at external environment, amplification human stem cell and hemopoietic stem cell family, this effective amplification is particularly useful for hemopoietic stem cell and early stage progenitor cell, this method can be used for specific clinical application, and can continue, regular or interim harvested cell.
2. according in the described device of claim 1, technical characterictic of the present invention is, a kind of bio-reactor of cultivating human stem cell and human hematopoietic stem that is exclusively used in is provided, by a sealing, the cultivation chamber that boundary is clear and definite is formed, in this chamber, human stem cell or hemopoietic stem cell can be induced and be cultivated, the semi-permeable membranes of being made of (1) gas-permeable, liquid-impermeable body following part suitably is placed on this chamber, thereby this chamber is divided into a gas chamber, cultivate chamber for one, in gas chamber, exchanging gas enters the cultivation chamber of opposite side by semi-permeable membranes; (2) the liquid cell culture fluid enters the cultivation chamber by the radial structure guiding inboard at inlet position, the nutrient solution that was used is then by the outer side system of the guiding of outlet position radial structure, therefore cultivating the perfusion passage that forms without hindrance, a radial nutrient solution between chamber import and the outlet; (3) partial structure is by the gas of above-mentioned gas chamber's perfusion cellular respiration; (4) partial structure is used for continuing, regularly or interimly gather in the crops non-adherent cell from cultivate chamber.
3. according in the described device of claim 1, bio-reactor provided by the invention is suitable for cultivates human stem cell or hemopoietic stem cell, and be waterproof at the semi-permeable membranes of this employing but can see through the film of gas, the bio-reactor of this suitable cultivation human stem cell or hemopoietic stem cell, its cell cultures chamber, the part that comprises suitable cell adhesion in a surface and growth is used for continuing with another, the part of regular or interim results non-adherent cell, back some is positioned at outlet, its guiding liquids cell cultures nutritive medium, move in the culture chamber indoor circulation, the structure of above-mentioned results non-adherent cell optionally harvested cell is based on the density of cell, the culture chamber chamber surface has a panel region cell adhesion, the above-mentioned suitable cell adhesion or the surf zone of attaching are that bioactivity surface is exclusively used in the specific cells adhesion, the above-mentioned suitable cell adhesion and the film of growth are suitable for the cell cultures chamber and place gas semi-permeable membranes top, cell culture chamber is indoor, constituting an obstruction liquid culture media mobile between the film of gas semi-permeable membranes and above-mentioned suitable cell adhesion/growth separates, the cell cultures chamber is the offside that is positioned at cell adhesion, and gas chamber is positioned at the offside that obstruction liquid culture media flows and separates.
4. according in the described device of claim 1, technical characterictic of the present invention is, human stem cell and human hematopoietic stem amplification system constitute (1) gas semi-permeable membranes by following part, in above-mentioned chamber, be divided into gas chamber and cultivate chamber, the exchanging gas in air chamber sees through the gas semi-permeable membranes and enters the cultivation chamber; (2) radial entrance structure and radial export structure form the passage of a radial liquid-flow; (3) dabbling cellular respiration gas enters the cultivation chamber by air chamber; (4) a part of structure can be gathered in the crops non-adherent cell, storage facility structure can retaining liquid shape cell culture media, first pump structure, can enter the cell cultures chamber by the perfusion fluid cell culture media, the source of cellular respiration gas, second pump structure can pour into cellular respiration gas by the cell cultures chamber.
5. according in the described device of claim 1, technical characterictic of the present invention is, the source of cellular respiration gas is a kind of compressed mixed gas, the source of cellular respiration gas is a kind of cultivation incubator, perfusion enters and comprises a kind of moisture in the cellular respiration gas, air is by humidification before being fed into the cultivation chamber, first kind of pump structure is the liquid filling pump, the fresh liquid cell culture media of this perfusion enters cell culture chamber, and extract used aqueous cell media out from the cell cultures chamber, cell adhesion/growth film is suitable for the cell cultures chamber, and be positioned at above the ventilative semi-permeable membranes, gas semi-permeable membranes and cell adhesion film are separated gas chamber and are cultivated chamber, and liquid media separate the offside that is positioned at cell adhesion/growth film, the cell cultures chamber comprises a kind of film of energy adhesion/one-tenth growing cells, the method that selectivity results non-adherent cell is selected based on cell density, sun hinders the mobile offside that is positioned at cell adhesion/growth film that separates of liquid culture media, with offside with air chamber, the gas semi-permeable membranes can see through gas but can not see through liquid, the used antibody of bio-reactor is for immature cell helps out, and above-mentioned antibody is CD34
+Antibody, above-mentioned antibody is distributed in around the gas semi-permeable membranes.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160434A (en) * | 2011-12-13 | 2013-06-19 | 汪华 | An automatic cell culture device capable of simulating internal environments of organisms |
| CN104870629A (en) * | 2012-10-26 | 2015-08-26 | 麻省理工学院 | Control of carbon dioxide levels and pH in small volume reactors |
| CN107208024A (en) * | 2015-01-20 | 2017-09-26 | 富士胶片株式会社 | Cell culture apparatus and cell culture processes |
| US10472602B2 (en) | 2012-10-26 | 2019-11-12 | Massachusetts Institute Of Technology | Humidity control in chemical reactors |
| US10479973B2 (en) | 2013-08-23 | 2019-11-19 | Massachuesetts Institute Of Technology | Small volume bioreactors with substantially constant working volumes and associated systems and methods |
| CN112567016A (en) * | 2018-08-20 | 2021-03-26 | 爱平世股份有限公司 | Cell culture device |
-
2005
- 2005-07-21 CN CN 200510028019 patent/CN1763173A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160434A (en) * | 2011-12-13 | 2013-06-19 | 汪华 | An automatic cell culture device capable of simulating internal environments of organisms |
| CN103160434B (en) * | 2011-12-13 | 2014-06-18 | 汪华 | An automatic cell culture device capable of simulating internal environments of organisms |
| CN104870629A (en) * | 2012-10-26 | 2015-08-26 | 麻省理工学院 | Control of carbon dioxide levels and pH in small volume reactors |
| US10472602B2 (en) | 2012-10-26 | 2019-11-12 | Massachusetts Institute Of Technology | Humidity control in chemical reactors |
| US11459538B2 (en) | 2012-10-26 | 2022-10-04 | Sanofi | Humidity control in chemical reactors |
| US11725176B2 (en) | 2012-10-26 | 2023-08-15 | Sanofi | Humidity control in chemical reactors |
| US10479973B2 (en) | 2013-08-23 | 2019-11-19 | Massachuesetts Institute Of Technology | Small volume bioreactors with substantially constant working volumes and associated systems and methods |
| US11827871B2 (en) | 2013-08-23 | 2023-11-28 | Massachusetts Institute Of Technology | Small volume bioreactors with substantially constant working volumes and associated systems and methods |
| CN107208024A (en) * | 2015-01-20 | 2017-09-26 | 富士胶片株式会社 | Cell culture apparatus and cell culture processes |
| CN112567016A (en) * | 2018-08-20 | 2021-03-26 | 爱平世股份有限公司 | Cell culture device |
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