CN100384987C - Method for expanding hemopoietic stem cell under three-dimensional condition - Google Patents
Method for expanding hemopoietic stem cell under three-dimensional condition Download PDFInfo
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Abstract
The present invention belongs to the field of biological technology and tissue engineering, which particularly relates to a two cell co-culture method under the three-dimensional condition. The method is characterized in that nurse cells are embedded into micro gel beads of calcium alginate; the embedded nurse cells and hemopoietic stem cells are co-cultured in a rotating wall type bioreactor to collect the hemopoietic stem cells and the nurse cells. The method has the advantages that micro gel bead channels enable important growth factors secreted by the nurse cells to penetrate through the hemopoietic stem cells outside a micro gel bead nourishing part; cells of two different sources are effectively separated from each other by the micro gel beads so that the action of immune isolation is performed, and the two kinds of cells are favorably separated and collected; and the rotating wall type bioreactor provides a three-dimensional suspension growing environment for the hemopoietic stem cells so that shearing force acting on the hemopoietic stem cells is effectively reduced, and the hemopoietic stem cells are favorably amplified.
Description
Technical field
The invention belongs to biotechnology and field of tissue engineering technology, particularly a kind of method of expanding hemopoietic stem cell under three-dimensional condition.
Background technology
Cell is in the growing environment complicated in the body, exist important contact between different types of cell, some cells in vitro is needing to rely on nourishing, the support effect of other cell to them when cultivating in varying degrees, and the former is called by nurse cell, and the latter is called nurse cell.This nourishing effect is particularly important for the vitro culture of stem cell, as the vitro culture of hemopoietic stem cell.
Hemopoietic stem cell has wide practical use clinically, it can rebuild patient behind the high dosage chemicotherapy hemopoietic system, carry out gene therapy and immunotherapy, the ripe blood products of preparation etc.But the quantity limitation problem that has hemopoietic stem cell in actual applications needs amplification on a large scale.The interior microenvironment of analogue body has been considered to the best approach of amplifying candidate stem cell in vitro at present.Stroma cell is the nurse cell that a class can play the nourishing effect to hemopoietic stem cell, and multiple stromal cell lines has been used to the amplification in vitro of hematopoiesis support stem cell.They can secrete multiple known, unidentified growth factor is kept and amplifying candidate stem cell, avoid hemopoietic stem cell in the vitro culture process gradually the differentiation and apoptosis.
On the means that realize two kinds of co-culture of cells, most of research has all adopted stroma cell and hemopoietic stem cell directly to contact cultured method altogether, promptly at first cultivating vessel bottom laying stroma cell layer, use the radiation exposure stroma cell of doses to suppress the activity of stroma cell then, again hemopoietic stem cell is cultivated on the stroma cell layer.The cultural method of this direct contact can support to nourish hemopoietic stem cell, promotes the amplification of hemopoietic stem cell, but the part hemopoietic stem cell is sticked on the stroma cell, is unfavorable for the separation and the results of two kinds of cells; If also there is the immunological rejection problem in allogenic two kinds of cells contacting.At present, before the hemopoietic stem cell of the vitro culture input human body, stroma cell must separate with hemopoietic stem cell fully, with the biological safety that guarantees to implant.Although the investigator who has uses nethike embrane of small-bore etc. that these the two kinds of cells in the co-culture system are separated, improved the problem of cellular segregation to a certain extent, as (Experimental Hematology, 27 such as Kawada, 904,1999) use a kind of porous-film with cord blood CD 34
+Cell and mouse stroma cell separate, but the fine hair of these two kinds of cells still can directly contact by the porous nethike embrane, and immune problem still exists.Therefore need to isolate in the new common cultivation of exploitation the method for allos cell.
In addition, the common cultivation of present most of stroma cells and hemopoietic stem cell all is to carry out under the culture condition of static state, as using orifice plate or culturing bottle.Although static cultivation is easy, there is following drawback:
1) nutrient solution lacks and effectively to mix and cause material such as nutritive ingredient, dissolved oxygen, meta-bolites such as cytokine and pH value to form concentration gradient in substratum;
2) the various parameters of static cultivation system generally all are difficult to on-line monitoring and control;
3) need manually change operation such as liquid, increase the danger of polluting;
4) two-dimensional static is cultivated away from the intravital three dimensional growth environment of people, easily causes the differentiation of ancestral cells to a certain extent.
Therefore, need the suitable Three-Dimensional Dynamic co-culture system of development, improve culture environment to prepare all guaranteed hemopoietic stem cell of quality and quantity.Through being usually used in the filling type (Palsson etc. that stroma cell and hemopoietic stem cell are cultivated altogether, Bio/Technology, 11,368,1993) and fixed-bed type bio-reactor (Meissner etc., Cytotechnology, 30,227,1999), because the hydrodynamic shear of their inside is bigger, stroma cell and hemopoietic stem cell interaction degree are limited, the poor effect of amplifying candidate stem cell.
Summary of the invention
The method that the purpose of this invention is to provide a kind of expanding hemopoietic stem cell under three-dimensional condition.
Technical scheme of the present invention may further comprise the steps:
1. nurse cell is provided.New zealand white rabbit around getting about age body weight 2.5kg, injection air method is put to death, and takes out femur and shin bone, is immersed in 75% the alcohol 30 minutes, is transferred in the aseptic super clean bench.Cut off the bone two ends with bone forceps, PBS washes medullary space, goes out marrow.Add the dilution of serum-free DMEM substratum, centrifugal 10 minutes of 1200rpm.Abandon lipid layer, add the dilution of serum-free DMEM substratum.Mix centrifugal 30 minutes of 3000rpm then by 1: 1 volume ratio with Ficoll cellular segregation liquid.Draw interfacial layer, add serum-free DMEM substratum and clean centrifugal 5 minutes of 1200rpm 2 times.Remove supernatant, add the DMEM substratum that contains 20% foetal calf serum, adjust cell concn, with 5 * 10
5Cellscm
-2Density be inoculated in the culturing bottle.Remove suspension cell after three days, per five and half amounts are changed liquid.When former generation the rabbit bone marrow mescenchymal stem cell reaching 80% at the bottom of the culturing bottle when merging, add concentration and be 0.05% pancreatin (containing 0.02%EDTA) digestion, in the cultivation of going down to posterity of 1: 3 ratio.
2. alginate calcium micro gel bead embedding nurse cell.Preparation density is 2.5 * 10
5CellsmL
-1The 4th generation the rabbit bone marrow mesenchyma stem cell suspension, mix with 1: 4 volume ratio with 1.5% sodium alginate soln, through 5
#Syringe needle dropwise is added drop-wise in 1.5% calcium chloride solution, constantly stirs, and react 10 minutes, obtains micro gel bead with the filtration of 20 eye mesh screens, and PBS washs three times, obtains diameter and be 2 millimeters the alginate calcium micro gel bead that is embedded with the rabbit bone marrow mescenchymal stem cell.The micro gel bead of preparation minor diameter will increase the use HV generator, its preparation process as shown in Figure 2, wherein, the diameter of alginate calcium micro gel bead mainly by needle point diameter, voltage, two interpole gaps from and sodium alginate stream of liquid droplets speed control.
3. separation of human Cord blood mononuclearcell.Cord blood is from healthy puerpera's term birth or mature surgical neonate, each former blood collection capacity average out to 50～100mL.Be 1.077gmL with density after the Cord blood collection
-1Ficoll cellular segregation liquid density gradient centrifugation after obtain mononuclearcell, the volume ratio of Ficoll parting liquid and Cord blood is 1: 1～1: 2.Twice (1000rpm, 5min), it is standby to adjust cell density with IMDM basic medium centrifuge washing for cell after the separation.
4. preparation rotating wall vessel bioreactor.(rotating wall vessel RWV) mainly is made of two circulation loops rotating wall vessel bioreactor: (1) substratum circulation loop; (2) gas circulation loop.Particularly, the substratum circulation is: substratum is delivered to oxygenation in the oxygenator 10 by peristaltic pump 11, enter culturing room 13 by RWV left end opening for feed afterwards and provide nutrient for cell, the substratum that oxygen level has reduced enters peristaltic pump 11 from RWV right-hand member discharge port, send into oxygenation in the oxygenator 10 again, the circulation of beginning next round.Gas circulation is: CO
2Contain 5%CO in the incubator 8
2The gas of 95% air is sent into by air pump 9 and is culture medium supplemented oxygen in the oxygenator 10, returns CO then
2In the incubator 8, constantly circulation.RWV main part oxygenator 10 is placed on CO with culturing room 13
2In the stay-warm case 15 outside the incubator 8.Before the experiment, after use clean-out system and soft brush clean each parts of RWV, ultrapure water flushing three times.Then each parts are immersed in 75% the spirituous solution and spend the night, again with ultrapure water flushing three times.Assemble the main part in the RWV stay-warm case 15, main part and the outer pipeline that connects, oxygenator are wrapped up respectively and autoclaving (121 ℃, 30min), dry for standby.
5. in rotating wall vessel bioreactor, the three-dimensional suspension cultivated the nurse cell and the hemopoietic stem cell of the embedding of alginate calcium micro gel bead altogether.Assembling RWV culture systems under the aseptic condition is filled with aseptic PBS circulation one hour in super clean bench, emits PBS, injects nutrient solution, and the temperature controlled switch of opening in the supervisory control desk 12 is controlled at 37 ℃ with temperature.With density is 2～5 * 10
5CellsmL
-1The human cord blood mononuclearcell and the nurse cell suspension inoculation of alginate calcium micro gel bead embedding to RWV culturing room 13, build stay-warm case 15, open peristaltic pump 11 and air pump 9.After one hour, open the power switch of adjustable-speed motor 14 in the supervisory control desk 12, make by adjusting knob that the inner/outer tube of RWV culturing room 13 is equidirectional to be rotated with circular frequency, this moment, total system was started working.The rotating speed of culturing room 13 progressively increases, and through 3rpm one hour, 5rpm 12 hours, finally reaches and remains on 6rpm, and cell and micro gel bead are suspended fully.
6. isolating hematopoietic stem cells and micro gel bead are gathered in the crops hemopoietic stem cell.After cultivating end altogether, with the suspension in the syringe extraction reactor culturing room 13, the 20 order stainless steel sifts that suspension drips behind high-temperature sterilization are online, and aseptic small beaker is placed in the screen cloth below.Micro gel bead is trapped within on the screen cloth, and the hemopoietic stem cell suspension then sees through screen cloth and enters in the beaker.With centrifugal 5 minutes of the hemopoietic stem cell suspension 1000rpm in the beaker, abandon supernatant, add 1 milliliter of nutrient solution, piping and druming, counting is seeded in the culturing bottle and cultivates.
7. the dissolving micro gel bead is gathered in the crops nurse cell.The micro gel bead of gathering in the crops is washed three times with PBS, be immersed in the sodium citrate solution that concentration is 55mM, reacted 10 minutes, centrifugal 10 minutes of 1500rpm abandons supernatant, adds 1 milliliter of nutrient solution again, piping and druming, meter mesenchymal stem cells MSCs number is seeded in the culturing bottle and cultivates.
Effect of the present invention and advantage are:
(1) can make nurse cell and hemopoietic stem cell under three-dimensional condition, carry out common cultivation, with of nourishing, the support effect of performance nurse cell to hemopoietic stem cell.
(2) two kinds of cells can keeping apart common cultivation help common separation and results of cultivating these two kinds of cells of back to avoid the immunological rejection effect between them.
(3) rotating wall vessel bioreactor has significantly reduced the shearing force of cell growing environment, cell is under the Three-Dimensional Dynamic condition all the time grows, and has improved the culture effect of cell.
(4) utilize the method for a kind of expanding hemopoietic stem cell under three-dimensional condition of the present invention, rabbit bone marrow mescenchymal stem cell and people's umbilical hemopoietic stem cell of alginate calcium micro gel bead embedding carried out dynamically cultivating altogether.The result shows that these two kinds of cells are equal well-grown in this culture system, karyocyte, CD34 after seven days
+(colony-forming unit-granulocyte macrophage, amplification times CFU-GM) can reach 170 times, 24 times and 14 times respectively for cell and grain monosystem progenitor cell.
Description of drawings
Fig. 1 is the method operational flowchart of a kind of expanding hemopoietic stem cell under three-dimensional condition of the present invention.
Fig. 2 is the synoptic diagram that the method for a kind of expanding hemopoietic stem cell under three-dimensional condition of the present invention prepares the alginate calcium micro gel bead.
Fig. 3 is that the method for a kind of expanding hemopoietic stem cell under three-dimensional condition of the present invention provides the rotary cell culture system process flow sheet of three-dimensional suspension growth environment for cell and micro gel bead.
Fig. 4 is that the method nurse cell and the hemopoietic stem cell of a kind of expanding hemopoietic stem cell under three-dimensional condition of the present invention cultivated synoptic diagram altogether.
Fig. 5 is under the method static conditions of a kind of expanding hemopoietic stem cell under three-dimensional condition of the present invention, and (A:Day 0 for the Photomicrograph of alginate calcium micro gel bead (diameter is 2 millimeters) embedding mouse neural stem cell; B:Day 9).
Fig. 6 is under the method static conditions of a kind of expanding hemopoietic stem cell under three-dimensional condition of the present invention, the osteoblastic Photomicrograph of alginate calcium micro gel bead (diameter is 2 millimeters) embedding mouse skull.
Fig. 7 is under the method static conditions of a kind of expanding hemopoietic stem cell under three-dimensional condition of the present invention, the Photomicrograph when alginate calcium micro gel bead of embedding rabbit bone marrow mescenchymal stem cell (diameter is 2 millimeters) and human cord blood hemopoietic stem cell are cultivated 168hr altogether (A:100 *; B:200 *).
Among the figure: 1. syringe; 2. beaker; 3. rotor; 4. iron stand; 5. anode; 6. negative electrode; 7. HV generator; 8.CO
2Incubator; 9. air pump; 10. oxygenator; 11. peristaltic pump; 12. supervisory control desk; 13. culturing room; 14. adjustable-speed motor; 15. stay-warm case; 16. the urceolus of rotary cell culture system culturing room; 17. the inner core of rotary cell culture system culturing room; 18. be embedded with the alginate calcium micro gel bead of nurse cell and the hemopoietic stem cell of the outer short-range interactions of micro gel bead; 19. the hemopoietic stem cell that micro gel bead is outer; 20. be embedded in the nurse cell in the micro gel bead.
Embodiment
Be described in detail specific embodiments of the invention below in conjunction with technical scheme and accompanying drawing.
Embodiment 1:
Present embodiment is alginate calcium micro gel bead embedding mouse neural stem cell vitro culture and evaluation.
For verify cell can be in the alginate calcium micro gel bead well growth, adopting diameter is 2 millimeters alginate calcium micro gel bead embedding mouse neural stem cell, carries out vitro culture and evaluation.Particularly, prepare the 6th generation mouse neural stem cell suspension, total cellular score is 9 * 10
5Individual, cell density is adjusted into 4 * 10
5CellsmL
-1This cell suspension and 1.5% sodium alginate soln mixed with 1: 4 volume ratio (this moment, cell density was 0.8 * 10
5CellsmL
-1), by 5
#Syringe needle dropwise is added drop-wise to 1.5%CaCl
2In the solution, agitation condition reacted 10 minutes down, and PBS washing three times uses screen filtration then to obtain micro gel bead.These micro gel beads are equally divided into three parts, are inoculated in the culturing bottle that 3 milliliters of nutrient solutions are housed and cultivate.Other gets the mouse neural stem cell suspension of a same total cellular score, is divided into three parts, without the micro gel bead embedding, with 1 * 10
5CellsmL
-1Density be inoculated in the culturing bottle that 3 milliliters of nutrient solutions are housed, group is carried out static cultivation in contrast.
Cultivate after 7 days, with 55mM sodium citrate solution dissolving alginate calcium micro gel bead, reacted 10 minutes, centrifugal 10 minutes of 1500rpm abandons supernatant, adds 1 milliliter of substratum, piping and druming, and counting, immunohistochemical methods is identified.Gather in the crops the neural stem cell of control group simultaneously, digestion, piping and druming, counting, immunohistochemical methods is identified.
More than test equal triplicate.
Experimental result: cultivate after 7 days, cellular control unit density reaches 2 * 10
5CellsmL
-1, amplification times is 2 times; Alginate calcium micro gel bead embedding group cell density reaches 1.58 * 10
5CellsmL
-1, amplification times is 1.98 times.Immunohistochemical methods identifies that the neural stem cell of finding in the micro gel bead is the same with the control group neural stem cell, can both express Nestin, and can neuralward unit and spongiocyte differentiation.Fig. 5 is that (A:Day 0 for the Photomicrograph of alginate calcium micro gel bead (diameter is 2 millimeters) embedding mouse neural stem cell; B:Day 9).
This embodiment explanation: cell can well be grown in the alginate calcium micro gel bead; Under the static culture conditions, cultivate with common cultured cells growing state in the micro gel bead roughly the same, simultaneous verification the mass-transfer performance of alginate calcium micro gel bead good; Because neural stem cell is very responsive to shearing force, prompting is under dynamic condition, and culturing cell may be bred more in the micro gel bead.
Embodiment 2:
Broken capsule yield rate when present embodiment is alginate calcium micro gel bead embedding mouse skull scleroblast cell.
The use diameter is 2 millimeters an alginate calcium micro gel bead embedding mouse skull scleroblast, determines the yield rate of cell by sodium citrate solution dissolving micro gel bead.Particularly, be 6 * 10 with 1 milliliter of density
5CellsmL
-1Mouse skull scleroblast and 4 milliliter of 1.5% sodium alginate soln mix, by 5
#Syringe needle dropwise is added drop-wise to 1.5%CaCl
2In the solution, agitation condition reacted 10 minutes down, PBS washing three times.Add 55mM sodium citrate solution effect 10 minutes, centrifugal 10 minutes of 1500rpm abandons supernatant, adds 1 milliliter of substratum, piping and druming, and counting calculates the cell harvesting rate.
More than test triplicate.
Experimental result: three times experiment gained cell harvesting rate is respectively 86.7%, 79.2%, 96.4%, and average yield rate reaches 87.4%.This embodiment explanation: it is feasible adopting the method for Trisodium Citrate dissolving micro gel bead harvested cell; After co-culture of cells, can gather in the crops most nurse cell by this method.Fig. 6 is the osteoblastic Photomicrograph of alginate calcium micro gel bead (diameter is 2 millimeters) embedding mouse skull.
Embodiment 3:
The cultivation of present embodiment behaviour umbilical cord blood hematopoietic stem cell in rotating wall vessel bioreactor.
In order to verify that the RWV culture systems can cultivate the more sensitive stem cell of growing environment, present embodiment end user umbilical cord blood hematopoietic stem cell is cultivated in the RWV culture systems.
The separation of human cord blood mononuclearcell: Cord blood is from healthy puerpera's term birth or mature surgical neonate, each former blood collection capacity average out to 50～100mL.Be 1.077gmL with density after the Cord blood collection
-1Ficoll lymphocyte separation medium density gradient centrifugation (2500r/min, 25min), centrifugal acquisition mononuclearcell (contains the CD34 about 1%
+Twice of IMDM basic medium centrifuge washing in back cell) (1000rpmin, 5min) standby.
The preparation of bio-reactor before the experiment: after using clean-out system and soft brush to clean each parts of RWV, ultrapure water flushing three times.Each parts are immersed in 75% the spirituous solution and spend the night, again with ultrapure water flushing three times.Assemble the main part of RWV, main part and the outer pipeline that connects, oxygenator are wrapped up respectively and autoclaving (121 ℃, 30min), dry for standby.
Inoculation and the cultivation of hemopoietic stem cell in reactor: open temperature controlled switch, RWV main part temperature in the stay-warm case is controlled at 37 ℃, the human cord blood mononuclearcell is with 2 * 10
5CellsmL
-1Density be inoculated in the RWV culturing room, open peristaltic pump and air pump.Nutrient solution is a basic medium with IMDM, adds 10% foetal calf serum, 10% horse serum and following somatomedin: SCF 16ngmL
-1, FL 7.47ngmL
-1, TPO7.47ngmL
-1, IL-35.33ngmL
-1, G-CSF 3.33ngmL
-1, GM-CSF 2.13ngmL
-1Culturing room's rotating speed of reactor is via 0rpm one hour, 3rpm one hour, and 5rpm remained on 6rpm after 12 hours.
Cultivated 7 days, take a sample in respect of the karyocyte number every day, and respectively at 0hr, 144hr and 197hr carry out CD34
+Cell flow cytometry analysis and colony form (colony-forming unit-granulocyte/macrophage, CFU-GM) ability detection.The karyocyte number meets or exceeds 1.5 * 10 in detecting culture system
6CellsmL
-1The time, extract the part cell suspension, add fresh medium simultaneously, make the karyocyte density in the system be no more than this numerical value.The cell that takes out is thought equally and is still grown in reactor, counts amplification times.
Cultivation results: three-dimensional suspension culture is after 7 days in the RWV culture systems for the human cord blood hemopoietic stem cell, and karyocyte has increased more than 400 times, CD34
+Cell amplification more than 30 times, the CFU-GM progenitor cells amplification more than 20 times.
This embodiment explanation: rotating wall vessel bioreactor can provide good three-dimensional suspension growth environment to hemopoietic stem cell, has promoted the amplification of hemopoietic stem cell; It is feasible using the outer culturing purposes cell of this reactor body.
Embodiment 4:
Present embodiment is the amplification in vitro of supporting, nourishing the human cord blood hemopoietic stem cell under the alginate calcium micro gel bead embedding rabbit bone marrow mescenchymal stem cell static conditions.
The effect that can play support to the hemopoietic stem cell of outside, nourish for the nurse cell of verifying the embedding of alginate calcium micro gel bead has been carried out common cultivation with the rabbit bone marrow mescenchymal stem cell and the human cord blood hemopoietic stem cell of alginate calcium micro gel bead embedding under the static conditions in six well culture plates.
The separation of rabbit bone marrow mescenchymal stem cell and cultivation: the new zealand white rabbit around getting about age body weight 2.5kg, injection air method is put to death, and takes out femur and shin bone, is immersed in 75% the alcohol 30 minutes, is transferred in the aseptic super clean bench.Cut off the bone two ends with bone forceps, PBS washes medullary space, goes out marrow.Add the dilution of serum-free DMEM substratum, centrifugal 10 minutes of 1200rpm.Abandon lipid layer, add the dilution of serum-free DMEM substratum.Mix centrifugal 30 minutes of 3000rpm then by 1: 1 volume ratio with Ficoll cellular segregation liquid.Draw interfacial layer, add serum-free DMEM substratum and clean centrifugal 5 minutes of 1200rpm 2 times.Remove supernatant, add the DMEM substratum that contains 20% foetal calf serum, adjust cell concn, with 5 * 10
5Cellscm
-2Density be inoculated in the culturing bottle.Remove suspension cell after three days, per five and half amounts are changed liquid.When former generation the rabbit bone marrow mescenchymal stem cell reaching 80% at the bottom of the culturing bottle when merging, add concentration and be 0.05% pancreatin (containing 0.02%EDTA) digestion, in the cultivation of going down to posterity of 1: 3 ratio.
Alginate calcium micro gel bead embedding rabbit bone marrow mescenchymal stem cell: preparation density is 2.5 * 10
5CellsmL
-1The 4th generation the rabbit bone marrow mesenchyma stem cell suspension, mix with 1: 4 volume ratio with 1.5% sodium alginate soln, through 5
#Syringe needle dropwise is added drop-wise in 1.5% calcium chloride solution, constantly stirs, and react 10 minutes, obtains micro gel bead with screen filtration, and PBS washs three times, obtains diameter and be 2 millimeters the alginate calcium micro gel bead that is embedded with the rabbit bone marrow mescenchymal stem cell.
The separation of human cord blood mononuclearcell: with embodiment 3.
Rabbit bone marrow mescenchymal stem cell and human cord blood mononuclearcell are cultivated altogether in six well culture plates: it is 3 * 10 that the micro gel bead of embedding rabbit bone marrow mescenchymal stem cell is allocated in 6 well culture plates with density by 30 in every hole
5CellsmL
-1The human cord blood mononuclearcell mix, put at 37 ℃ 5%CO
2Cultivate in the incubator.Nutrient solution is basic medium with IMDM, adds 20%FBS, and replenishes the somatomedin of following low dosage, SCF 16ngmL
-1, FL 7.47ngmL
-1, TPO 7.47ngmL
-1, IL-3 5.33ngmL
-1, G-CSF3.33ngmL
-1, GM-CSF 2.13ngmL
-1
The rabbit bone marrow mescenchymal stem cell of micro gel bead embedding is about 4: 100 with the ratio of the outer human cord blood mononuclearcell quantity of micro gel bead when initial, i.e. mescenchymal stem cell and CD34
+The ratio of cell is about 4: 1 (CD34 when initial
+Cell account for mononuclearcell 1%), the 2mL nutrient solution is added in every hole.Change liquid 10% every day, in respect of karyocyte density, and respectively at 0hr, 72hr, 120hr and 168hr carry out CD34
+Cell flow cytometry analysis and CFU-GM colony detect.
Cultivation results: in seven days culturing process, karyocyte, CD34 in the co-culture system
+Cell and CFU-GM amplification times reach 9 times, 20 times and 9 times respectively.The Photomicrograph of Fig. 7 when co-culture system is cultured to 168hr for this reason (A:100 *; B:200 *).
This embodiment explanation: the alginate calcium micro gel bead is a kind of effective co-culture of cells means, can play immune isolated effect, and helps common the cultivation separating of back mescenchymal stem cell and hemopoietic stem cell; The rabbit bone marrow mescenchymal stem cell that is embedded in the micro gel bead can the amplification of backer's umbilical cord blood hematopoietic ancestral cells effectively under external static conditions, illustrates to use micro gel bead embedding nurse cell not hinder this nourishing effect; If cooperate the three-dimensional suspension growth environment of RWV culture systems, the amplification in vitro of hemopoietic stem cell can be supported, be nourished to micro gel bead embedding nurse cell better.
Embodiment 5:
Present embodiment is the amplification in vitro of supporting, nourishing the human cord blood hemopoietic stem cell under the alginate calcium micro gel bead embedding rabbit bone marrow mescenchymal stem cell dynamic condition.
The effect that can play support to the hemopoietic stem cell of outside, nourish for the nurse cell of verifying the embedding of alginate calcium micro gel bead has been carried out common cultivation with the rabbit bone marrow mescenchymal stem cell and the human cord blood hemopoietic stem cell of alginate calcium micro gel bead embedding under the dynamic condition in rotating wall vessel bioreactor.
The separation of rabbit bone marrow mescenchymal stem cell and cultivation: with embodiment 4.
Alginate calcium micro gel bead embedding rabbit bone marrow mescenchymal stem cell: with embodiment 4.
The separation of human cord blood mononuclearcell: with embodiment 3.
The preparation of bio-reactor before the experiment: with embodiment 3.
The rabbit bone marrow mescenchymal stem cell is cultivated altogether with the human cord blood mononuclearcell in the rotating wall vessel bioreactor: opening the interior RWV main part temperature of temperature controlled switch control stay-warm case at 37 ℃, is 3 * 10 with the alginate calcium micro gel bead and the density of 450 embedding rabbit bone marrow mescenchymal stem cells
5CellsmL
-1The human cord blood mononuclearcell is inoculated in the RWV culturing room, opens peristaltic pump and air pump.Nutrient solution is a basic medium with IMDM, adds following somatomedin: SCF 16ngmL
-1, FL 7.47ngmL
-1, TPO 7.47ngmL
-1, IL-35.33ngmL
-1, G-CSF 3.33ngmL
-1, GM-CSF 2.13ngmL
-1Culturing room's rotating speed of reactor is via 0rpm one hour, 3rpm one hour, and 5rpm remained on 6rpm after 12 hours.
Cultivated 7 days, take a sample in respect of the karyocyte number every day, and respectively at 0hr, 144hr and 197hr carry out CD34
+Cell flow cytometry analysis and CFU-GM detect.The karyocyte number meets or exceeds 1.5 * 10 in detecting culture system
6CellsmL
-1The time, extract the part cell suspension, add fresh medium simultaneously, make the karyocyte density in the system be no more than this numerical value.The cell that takes out is thought equally and is still grown in reactor, counts amplification times.After cultivating end altogether, separate hematopoietic cell and micro gel bead.Micro gel bead with embedding rabbit bone marrow mescenchymal stem cell washes three times with PBS again, is immersed in the sodium citrate solution of 55mM, reacts 10 minutes, and results rabbit bone marrow mescenchymal stem cell is seeded in the culturing bottle and cultivates.
Cultivation results: in seven days culturing process, karyocyte, CD34 in the co-culture system
+Cell and CFU-GM amplification times reach 170 times, 24 times and 14 times respectively.The broken capsule yield rate of nurse cell---rabbit bone marrow mescenchymal stem cell reaches about 90%.
This embodiment explanation: in rotating wall vessel bioreactor, be embedded in the amplification in vitro that rabbit mescenchymal stem cell in the alginate calcium micro gel bead had nourished, supported hemopoietic stem cell effectively.Although the amplification times of hemopoietic stem cell is not as good as the height among the embodiment 3 in the present embodiment, use the embedding nurse cell to substitute animal serum in the present embodiment, near clinical, have medical value more more.
Although the present invention is that the hemopoietic stem cell that nourishes the human cord blood source with the rabbit bone marrow mescenchymal stem cell is that example is described, this description and not meaning that is construed as limiting the present invention.With reference to description of the invention, the nurse cell of other kind, by nurse cell and embodiment other the distortion, all can expect for those skilled in the art.Therefore, such distortion can not break away from affiliated claim restricted portion and spirit.
Claims (1)
1. the method for an expanding hemopoietic stem cell under three-dimensional condition is characterized in that following steps:
(1) provide nurse cell: nurse cell is rabbit femoral and tibial bone bone marrow-drived mesenchymal stem, with 5 * 10
5Cellscm
-2Density be inoculated in the DMEM substratum that contains 20% foetal calf serum and cultivate, when cell is reaching 70% when merging at the bottom of the culturing bottle, adding and containing 0.02%EDTA concentration is 0.05% tryptic digestion, carries out the routine cultivation of going down to posterity in 1: 3 ratio;
(2) alginate calcium micro gel bead embedding nurse cell: adopting 1.5% sodium alginate soln and density is 2.5 * 10
5CellsmL
-1The nurse cell suspension mix with 4: 1 volume ratios after, dropwise drop in 1.5% calcium chloride solution through the 5# syringe needle, react 10 minutes, it is 2 millimeters the alginate calcium micro gel bead that is embedded with nurse cell that the filtration of 20 eye mesh screens makes diameter;
(3) separation of human Cord blood mononuclearcell: Cord blood is from healthy puerpera's term birth and mature surgical neonate, each former blood collection capacity average out to 50～100mL, the density gradient centrifugation separation obtains mononuclearcell, with IMDM basic medium 1000rpm, 5min centrifuge washing twice, it is standby to adjust cell density;
(4) prepare rotating wall vessel bioreactor: rotating wall vessel bioreactor mainly is made of substratum circulation loop and two circulation loops of gas circulation loop; The substratum circulation loop comprises peristaltic pump, oxygenator, culturing room, substratum is delivered to oxygenation in the oxygenator by peristaltic pump, enter culturing room by bio-reactor left end opening for feed afterwards and provide nutrient for cell, the substratum that oxygen level has reduced enters peristaltic pump from bio-reactor right-hand member discharge port, send into oxygenation in the oxygenator again, the circulation of beginning next round; Gas circulation loop comprises CO
2Incubator, air pump, oxygenator, CO
2Contain 5%CO in the incubator
2With the gas of 95% air, being sent in the oxygenator by air pump is culture medium supplemented oxygen, returns CO then
2In the incubator, constantly circulation; The oxygenator and the culturing room of this rotating wall vessel bioreactor main part are placed on CO
2In the stay-warm case outside the incubator,
(5) in rotating wall vessel bioreactor, the three-dimensional suspension cultivated the nurse cell and the hemopoietic stem cell of the embedding of alginate calcium micro gel bead altogether: the inner/outer tube of rotating wall vessel bioreactor culturing room is equidirectional to be rotated with circular frequency, rotating speed progressively increases from static, through 0rpm one hour, 3rpm one hour and 5rpm after 12 hours, finally reach and remain on 6rpm; The inoculum density of human cord blood mononuclearcell in rotating wall vessel bioreactor culturing room is 2～5 * 10
5CellsmL
-1
(6) isolating hematopoietic stem cells and micro gel bead, results hemopoietic stem cell: adopt 20 order stainless steel meshs to hold back the alginate calcium micro gel bead of embedding mesenchymal stem cells MSCs, collect filtered solution---hemopoietic stem cell suspension, centrifugal 5 minutes of 1000rpm, results hemopoietic stem cell;
(7) dissolving micro gel bead, the results nurse cell: adopting concentration is the alginate calcium micro gel bead 10 minutes that the sodium citrate solution dissolving of 55mM is embedded with nurse cell, centrifugal 10 minutes of 1500rpm, results nurse cell.
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| CN102465112A (en) * | 2010-11-01 | 2012-05-23 | 张正前 | Human umbilical cord blood hematopoietic stem cell high-efficiency in vitro amplification technology |
| CN102965330B (en) * | 2011-09-01 | 2014-07-09 | 中国科学院大连化学物理研究所 | Method for synergistic growth of multiple cells |
| CN102747029B (en) * | 2012-07-30 | 2013-09-25 | 何伟 | Culture method for retina progenitor cells and culture medium thereof |
| CN103122348A (en) * | 2012-09-20 | 2013-05-29 | 天津卫凯生物工程有限公司 | Islet microcapsules, islet model, preparation method and application |
| CN103881908A (en) * | 2012-12-20 | 2014-06-25 | 中国科学院大连化学物理研究所 | Bioreactor system for cell co-cultivation |
| CN104667348B (en) * | 2015-02-04 | 2016-03-02 | 北京大学第三医院 | A kind of Pharmaceutical composition containing sodium alginate and preparation method thereof |
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