CN115074320B - Preparation system and preparation method of high-concentration mesenchymal stem cell exosome in 3D culture environment - Google Patents
Preparation system and preparation method of high-concentration mesenchymal stem cell exosome in 3D culture environment Download PDFInfo
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Abstract
The invention provides a preparation system and a preparation method of a high-concentration mesenchymal stem cell exosome in a 3D culture environment, and relates to exosome production technologyDomain. The invention relates to 3D culture equipment of mesenchymal stem cells and a RoosterNourish TM MSC-XF and roosterCollet-EV Pro TM The organic combination can meet the long-time continuous production of the mesenchymal stem cells, and the obtained exosomes have high concentration and few impurities, thereby realizing the industrial production of the mesenchymal stem cell exosomes.
Description
Technical Field
The invention belongs to the technical field of exosome production, and particularly relates to a preparation system and a preparation method of a high-concentration mesenchymal stem cell exosome in a 3D (three-dimensional) culture environment.
Background
Exosomes are small membrane vesicles secreted by a variety of cell types, playing an important role in cell-to-cell communication. They can be used as a very valuable clinical tool for early diagnosis, prognostic evaluation and targeted therapy. The clinical use of exosomes is still in an early stage, with commercial viability due to the potential to apply cell and gene therapies to a large population.
Most developers of exosome industry related upstream and downstream innovative technologies are in preclinical or early clinical stages, demonstrating the safety and effectiveness of exosome-based therapies. From a manufacturing point of view, challenges exist in the production and characterization of exosomes. All problems with the transfer of modern technology will be in the large scale development, especially in the clinically relevant exosome manufacturing processes. This includes all steps and quality control procedures involved from laboratory scale production to commercial scale production. The objectives include enhanced process control, creation of as many closed jobs as possible, turning to more automated and digital processes, adoption of single-use systems, and implementation of as many field monitoring devices and sample analyses as possible.
The current mass production methods of exosomes mainly comprise: cell expansion is performed using tens of large (e.g., T-225) cell culture flasks, multiple multi-layer stacked array cell culture flasks, large fixed bed bioreactors, microcarriers or continuous production perfusion reactors. The composition and activity of exosomes reflect the physiological state of the cell at the time of secretion, irrespective of physiological or in vivo conditions. However, current mass production has common limitations, especially inefficiency. And when in production, the serum contains a large amount of polluted endogenous exosomes, a serum-free culture medium is required for cell expansion, and is limited by the factor of expansion production cost, and a high-sugar culture medium becomes the first choice for mesenchymal stem cell expansion, but can accelerate cell aging, so that exosome components are inconsistent in the long-term cell culture process. Therefore, how to improve the production efficiency of exosomes and balance the contradiction between serum in high-sugar culture medium is the research direction at present.
Development of exosomes as therapeutic agents requires GMP-compliant production and purification methods. 3D culture systems, serum-free media, and suitable for GMP-grade biological agents. Here, a process for preparing high-concentration low-impurity mesenchymal stem cell exosomes stable for a long period of time under a 3D culture environment based on a hollow fiber bioreactor (Hollow Fiber Bioreactor) and Rooster Bio mesenchymal stem cell expansion medium and Rooster Bio exosome secretion-promoting medium is described.
Disclosure of Invention
In view of the above, the present invention aims to provide a preparation system and a preparation method for high-concentration mesenchymal stem cell exosomes in a 3D culture environment, which adopt a 3D culture system, a serum-free medium and a biological agent suitable for GMP-grade, and are suitable for preparing high-concentration low-impurity mesenchymal stem cell exosomes stably for a long period of time.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation system of high-concentration mesenchymal stem cell exosomes in a 3D culture environment, which comprises 3D culture equipment of mesenchymal stem cells, a RoosterNourish TM MSC-XF medium and RoosterCollet-EV Pro TM A culture medium.
Preferably, the 3D culture apparatus of mesenchymal stem cells comprises a hollow fiber bioreactor or a fully automatic mesenchymal stem cell culture apparatus of the same principle as the hollow fiber bioreactor.
Preferably, the roosternourise TM MSC-XF medium includes RossterBasal TM MSC-XF basal medium and roosterNourish TM -MSCXF complete medium;
the RoosterNourish TM MSC-XF was fully cultured as in the RoosterBasal TM Addition of roosterboost to MSC-XF basal medium TM MSC-XF additive.
The invention also provides a preparation method of the high-concentration mesenchymal stem cell exosome based on the preparation system, which comprises the following steps: (1) By roosterNourish TM Performing cell expansion on the mesenchymal stem cells by using MSC-XF complete medium, and respectively collecting cells and cell culture solution supernatants when the cells grow to 85% fusion degree;
(2) After resuspension of the collected cells using the cell culture supernatant of step (1), the cells were isolated in a roosternourise TM -in an MSC-XF complete medium environment, inoculating into a 3D culture device of pretreated mesenchymal stem cells, performing medium replacement according to the consumption of glucose in the medium;
the first time a new roosternourise was exchanged when the glucose concentration was 60% depleted TM MSC-XF complete medium;
a second replacement of the new roosterNourish when the glucose concentration was depleted by 50% TM MSC-XF complete medium;
when the glucose concentration was depleted by 50%, a third replacement of the new RoosterCollect-EV Pro was performed TM Complete medium; the roosterColllect TM MSC-XF complete medium was at roosterCollect-EV Pro TM EV Pro was added to the medium TM Exosome secretion enhancers;
starting from the third medium change, cell supernatants were collected every 2 days, and mesenchymal stem cell exosomes were extracted using the cell supernatants.
Preferably, in step (1), the ratio of the components is 1×10 6 cell/mL inoculum size, mesenchymal stem cells were inoculated into the RosterNourish TM Cell expansion was performed in MSC-XF complete medium, and on day 4, the new RossterNourish was replaced after washing the cells with D-PBS TM MSC-XF complete medium, cell and cell culture supernatant were collected separately, based on cell growth to 85% confluence.
Preferably, the method comprises the steps of,the pretreatment of step (2) comprises a first circulation of PBS solution in the hollow fiber bioreactor for 48 hours, and a replacement of the RossterBasal TM MSC-XF basal medium was second cycled for 48 hours and replaced with RosterNourish TM MSC-XF complete medium third cycle 48h.
Preferably, after the third cycle, further comprising a new RosterNourish switch TM MSC-XF complete medium, cell seeding.
Preferably, the initial flow rate 25 is set after inoculation in step (2), the flow rate is increased to 28 after the first update, the flow rate is increased to 30 after the second update, and the flow rate is maintained 30 after the third update.
Preferably, after the third update in step (2), further comprising culturing for 34 days, the cell supernatant is collected every two days.
Preferably, after collecting the cell supernatant, further comprising centrifugation at 3500 Xg for 60min at 4℃and collecting the supernatant.
The beneficial effects are that: the invention provides a preparation system of high-concentration mesenchymal stem cell exosomes, wherein 3D culture equipment of the mesenchymal stem cells can meet the requirement of high-density long-term culture of a large number of cells under a three-dimensional physiological condition, and support the collection and concentration of exosomes under the culture condition of a cell growth period, and the collection of secretion products can maintain continuous production for a plurality of weeks or months; the RoosterNourish TM MSC-XF is a friendly, heterologous (XF) -free high sugar media for bioengineering, supporting the use of the RosterBooster TM MSC-XF additives, in vitro amplification of hMSCs can be performed in 2D or 3D biological environments. RoosterCollect-EV Pro TM Is a low-particle engineering Extracellular Vesicle (EV) collection kit which can improve the total yield of hMSC-EV.
Meanwhile, roosterNourish TM The MSC-XF complete culture medium is a culture medium without exosome pollution, does not cause vesicle pollution of the final product, can carry out mass expansion of mesenchymal stem cells in a short time, and simultaneously maintains the proliferation state of the cells. When the cells reach the high cell density of the fiber cell system, the cells can be directly replaced by the RoostterCollet-EV Pro TM CulturingThe substrate, during which no other solution substitutions are required. When exchanged for roosterCollect-EV Pro TM After the culture medium, the cells are not proliferated, but are maintained in a high vesicle secretion state, and the cells are not aged in a long-term high-sugar culture environment. Since the parent cell is in a non-proliferative state during exosome production, the exosomes secreted by the parent cell are also relatively stable.
Drawings
FIG. 1 is a fiber cell exosome production system;
FIG. 2 shows the pre-inoculation cell viability of three different cord tissue-derived mesenchymal stem cells for the P5 generation; FIG. 2a shows the pre-inoculation cell viability of the hUC-MSC-0901P5 generation, FIG. 2b shows the pre-inoculation cell viability of the hUC-MSC-0214P5 generation, and FIG. 2c shows the pre-inoculation cell viability of the hUC-MSC-1103P5 generation;
FIG. 3 shows that the concentration of exosomes harvested at different time points during the long-term culture of hUC-MSC-0214 was stable;
FIG. 4 shows that the grain size of the exosomes harvested at different time points during the long-term culture of hUC-MSC-0214 is stable;
FIG. 5 is a graph showing analysis of exosome subpopulations at different time points of nanoView;
FIG. 6 shows the exosome size analysis at various time points for nanoView;
FIG. 7 is a TERUMOCELL EXPANSION SYSTEM system.
Detailed Description
The invention provides a preparation system of high-concentration mesenchymal stem cell exosomes, which comprises a 3D culture device of mesenchymal stem cells, a roosterNourish TM MSC-XF medium and RoosterCollet-EV Pro TM A culture medium.
The 3D culture apparatus of mesenchymal stem cells of the present invention preferably comprises a hollow fiber bioreactor (Hollow fiber bioreactor, HFBR) or a full-automatic mesenchymal stem cell culture apparatus of the same principle as the hollow fiber bioreactor. The source of the 3D culture apparatus is not particularly limited in the present invention, and preferably includes a fiber cell company hollow fiber bioreactor, or TERUMO full-automatic mesenchymal stem cell culture equipment based on principleCELL EXPANSION SYSTEM (FIG. 7) the example is described by way of example in relation to a hollow fiber bioreactor from fiber cell, but this should not be construed as limiting the scope of the invention.
The HFBR of the present invention can collect and concentrate exosomes under cell growth phase culture conditions: (1) HFBR is the most similar method to the in vivo environment to culture a large number of cells and obtain their secretions. Can support a large number of cells to be cultured for a long time under the three-dimensional physiological condition at high density; (2) Molecular weight cutoff of the fiber allows nutrients and waste to pass through the fiber, but concentrates larger secreted products and exosomes in a small volume (capillary outside of the fiber tube), initially concentrating exosomes; (3) If serum is required for production, it can only be used to circulate the medium, while the extracellular space of the fiber tube containing cells and secreted exosomes can be kept serum-free. HFBR may facilitate the use of protein free media; cells are also bound to the porous scaffold and thus do not need to divide, cells grow freely after fusion, and collection of secreted products can sustain continuous production for weeks or months. The FiberCell hollow fiber bioreactor employed in the embodiments of the present invention can produce large amounts of highly concentrated extracellular vesicles in a space and time efficient manner. Primary Mesenchymal Stromal Cells (MSCs) can be cultured continuously at high density in this system. The current throughput is 10-to 40-fold compared to static bottle batch processing. Higher EV purity allows for easier periodic collection of cellular secretions to avoid starvation stress. The hollow fiber bioreactor culture conditions obtained a more in vivo cellular microenvironment and corresponding EV proteome/miRNA profile.
RoosterNourish according to the invention TM The MSC-XF medium preferably comprises RossterBasal TM MSC-XF basal medium and roosterNourish TM MSC-XF complete medium; the RoosterNourish TM Complete culture of MSC-XF is preferably performed in the RossterBasal TM Addition of roosterboost to MSC-XF basal medium TM -MSC-XF additive, and the addition ratio is preferably 500mL:10mL. RoosterNourish according to the invention TM MSC-XF is a friendly, heterologous (XF-free) bioengineering medium that eliminates medium replacement during rapid expansion of human mesenchymal stem cells/stromal cells (hMSCs) from a variety of tissue sources. The medium supports the use of RoosterReplenish TM MSC-XF additives, in vitro amplification of hMSCs can be performed in 2D or 3D biological environments. Roosternourise, compared to other MSC media TM MSC-XF has an unparalleled productivity, producing millions of cells per liter of medium consumed. With XF roosterVial TM MSC used together, 10 XhMSC can be extended in 4-6 days. Meanwhile, the culture medium is a high-sugar culture medium, so that the time cost of the cell expansion stage is greatly reduced, and the production cost of the cell expansion stage is reduced.
The invention relates to a RoostterCollet-EV Pro TM The medium preferably comprises roosterCollet-EV Pro TM Basal medium and roosterCollect-EV Pro TM Complete medium, and the RoosterCollect TM MSC-XF complete medium is preferably at the RooterCollet-EV Pro TM EV Pro was added to the medium TM Exosome secretion enhancers, more preferably at a ratio of 500mL:10mL. The invention relates to a RoostterCollet-EV Pro TM The culture medium is a low-particle, engineered Extracellular Vesicles (EV) collection kit, which can improve the total yield of hMSC-EV. After cell expansion, this medium can support cell health during hMSC-based EV collection, extend exosome collection window, increase EV yield, and reduce variability in 3D bioreactors and 2D bottle culture systems.
In the present invention, the above two media are combined, first, roosternourise TM MSC-XF is a culture medium free of exosome contamination, does not cause vesicle contamination of the final product, and can perform mass expansion of mesenchymal stem cells in a short time while maintaining the proliferation state of the cells. When the cells reach the high cell density of the fiber cell system, the cells can be directly replaced by the RoostterCollet-EV Pro TM The culture medium is not required to be put in any other solutionAnd (5) changing. When exchanged for roosterCollect-EV Pro TM After the culture medium, the cells are not proliferated, but are maintained in a high vesicle secretion state, and the cells are not aged in a long-term high-sugar culture environment. Since the parent cell is in a non-proliferative state during exosome production, the exosomes secreted by the parent cell are also relatively stable.
The invention also provides a preparation method of the high-concentration mesenchymal stem cell exosome based on the preparation system, which comprises the following steps: (1) By roosterNourish TM Performing cell expansion on the mesenchymal stem cells by using an MSC-XF culture medium, and respectively collecting cells and cell culture solution supernatants when the cells grow to 85% fusion degree;
(2) After resuspension of the collected cells using the cell culture supernatant of step (1), the cells were isolated in a roosternourise TM -in an MSC-XF complete medium environment, inoculating into a 3D culture device of pretreated mesenchymal stem cells, performing medium replacement according to the consumption of glucose in the medium;
the first time a new roosternourise was exchanged when the glucose concentration was 60% depleted TM MSC-XF complete medium;
a second replacement of the new roosterNourish when the glucose concentration was depleted by 50% TM MSC-XF complete medium;
when the glucose concentration was depleted by 50%, a third replacement of the new RoosterCollect-EV Pro was performed TM Complete medium;
starting from the third medium change, cell supernatants were collected every 2 days, and mesenchymal stem cell exosomes were extracted using the cell supernatants.
The invention utilizes roosterNourish TM Cell expansion of mesenchymal stem cells was performed on the MSC-XF medium, and cells were grown to 85% confluence, and cell culture supernatants were collected, respectively. The invention is preferably carried out in a way of 1X 10 6 cell/mL inoculum size, mesenchymal stem cells were inoculated into the RosterNourish TM Cell expansion was performed in MSC-XF complete medium, and on day 4, the new RossterNourish was replaced after washing the cells with D-PBS TM MSC-XF medium, collected separately based on cell growth to 85% confluenceCells and cell culture supernatants. The inoculation according to the invention preferably comprises inoculation into T-175 flasks and at 37℃with 5% CO 2 Is subjected to cell expansion under the conditions of (a). The mesenchymal stem cells of the present invention are preferably cells within the generation P10, and the generation P4 is exemplified in the examples, but they are not to be construed as the full scope of the present invention.
The invention preferably uses D-PBS to wash cells 2 times after the inoculation and then changes a new culture medium, the cells are collected for cell counting on the 6 th day (the cells grow to 85% fusion degree), cell quality detection comprises aseptic detection and cell phenotype detection, 50mL of cell culture supernatant is collected at the same time, the cell culture supernatant is filtered into a new centrifuge tube through a 0.22 mu m filter membrane by using a syringe, and the cell culture supernatant is preserved at 37 ℃ for standby.
After collecting the cells and cell culture supernatant, the invention uses the cell culture supernatant of step (1) to re-suspend the collected cells, and then uses the method of the invention in a RossterNourish TM In an MSC-XF complete medium environment, inoculating into a 3D culture device of the pretreated mesenchymal stem cells, and performing medium replacement according to the consumption of glucose in the medium.
The pretreatment according to the invention preferably comprises: removing residual substances in the manufacturing process, ensuring that the system cannot leak, confirming the sterile state of the system, and establishing the system into an environment suitable for cell growth; more preferably, the method comprises: in the hollow fiber bioreactor, the RoosterBasal was replaced by a first circulation of PBS solution for 48h TM MSC-XF basal medium was second cycled for 48 hours and replaced with RosterNourish TM MSC-XF complete medium was third circulated for 48h, and finally replaced with fresh RosterNourish TM MSC-XF complete medium, cells can be inoculated (FIG. 1). In the embodiment of the invention, when the pretreatment is carried out, the material is preferably sterilized, for example, the position of a stainless steel tube of a liquid storage bottle cap is adjusted, the mouth of the stainless steel tube is ensured to be below a 125mL scale, but the mouth of the stainless steel tube does not touch the bottom of a culture bottle, an outer layer bag of a culture cylinder is disassembled, and 2 liquid storage bottle connecting tubes in the culture cylinder are taken out; and respectively sleeving the bottle cap connecting pipes with the top openings of the stainless steel pipes, sealing the openings at the upper and lower ends of the stainless steel pipes by tinfoil paper, placing the stainless steel pipes into a sterilizing bag,together with the liquid storage bottle.
The pretreatment according to the invention preferably further comprises equipment assembly and PBS pretreatment in an ultra clean bench: (1) preparation of materials: a culture cylinder with an undetached inner bag, 150 mL centrifuge tube, 1 sterile threaded syringe (20 mL), sterilized liquid storage bottle and bottle cap, 250mL PBS and alcohol cotton ball (for disinfection);
(2) Pouring about 40mL of PBS into a 50mL centrifuge tube (the outside space of the fiber is filled later), pouring the rest PBS into a liquid storage bottle, filling the sterilized bottle cap into the liquid storage bottle, and screwing the cap;
(3) Connecting two silica gel hoses of the culture cylinder with bottle cap connecting pipes, sterilizing each joint by alcohol, and connecting the two hoses with the bottle cap connecting pipes slightly reversely, so as to avoid the pipeline being in a twisted state after the joint and ensure the tight joint;
(4) Confirming that two valves of the intra-fiber pipeline are opened, closing the valve of the extra-fiber space, manually pressing the one-way valve, injecting PBS into the intra-fiber pipeline from the liquid storage bottle, and closing the valve of the intra-fiber pipeline after bubbles are seen to emerge, wherein the pipeline is proved to be smooth;
(5) The syringe was used to aspirate 50mL of PBS, sterilize and open the two valves of the extrafibrous space. The syringe was screwed into the left outer opening and the PBS was slowly pumped into the syringe until the extra-fibrous space was filled, taking care not to spill the PBS out of the right outer opening.
(6) The valve of the external space of the fiber is closed, the valve of the internal pipeline of the fiber is opened to ensure the liquid circulation of the internal pipeline, the culture cylinder is loaded on the double-head pump, the flow rate is set at 20, and the culture cylinder is put into an incubator for circulation for 48 hours.
(7) After 48 hours of cycling, the cycle was replaced with a RoosterBasal TM MSC-XF basal medium was circulated for 48 hours.
The flow rate is set in the invention, because the equipment is provided with a matched pump head, the flow rate is controlled to be 1-30, and the flow rate is accelerated along with the higher sugar consumption (the more the cell quantity); the initial flow rate of the middle-sized barrels (C2008 and C2011) is recommended to be 20 (about 80-90 ml/min), and can be adjusted to 25 after 3-4 days of gradual increase of sugar consumption. The initial flow rate of the large cartridges (C2003 and C2018) was also recommended to be set at 20, and after 2 days of initial sugar consumption could be adjusted to 25-30.
The invention is used for replacing the roosterBasal TM Before the circulation of the MSC-XF basal medium, it preferably further comprises: (1) material preparation: 2 sterile threaded port syringe, 250mL roosterBasal TM MSC-XF basal medium, alcohol (for disinfection);
(2) sucking 30mL of basal culture medium into a needle cylinder for standby;
(3) sterilizing the bottle mouth, replacing the PBS liquid storage bottle with a liquid storage bottle containing a culture medium, manually pressing a one-way valve, injecting a basic culture medium, and ensuring that a pipeline is smooth;
(4) closing the inner pipeline valve in the fiber and opening the outer pipeline valve in the fiber;
(5) sucking out PBS from the left outer opening by using an empty syringe, screwing a new empty syringe at the right outer opening, connecting the left outer opening with a syringe containing 30mL of basic culture medium, injecting the culture medium into the culture cylinder, and slowly pushing in the process to avoid generating bubbles;
(6) closing the valve of the external space of the fiber, opening the valve of the internal pipe of the fiber, loading the culture cylinder on the double-head pump, setting the flow rate to be 25, putting the culture cylinder into an incubator for circulation for 48 hours, and replacing the culture cylinder with the RooterNourish TM MSC-XF complete medium.
The invention changes the roosterNourish TM Before the MSC-XF complete medium is circulated, it preferably further comprises: (a) preparation of materials: 2 sterile threaded needle cylinder, 250mL RoosterNourish TM MSC-XF complete medium, alcohol (for sterilization);
(b) Sucking 30mL of complete culture medium into a syringe for standby;
(c) Sterilizing the bottle mouth, replacing the basal medium liquid storage bottle with a liquid storage bottle containing the complete medium, manually pressing a one-way valve, injecting the complete medium, and ensuring that a pipeline is smooth;
(d) Closing the inner pipeline valve in the fiber and opening the outer pipeline valve in the fiber;
(e) Sucking out the basic culture medium from the left outer opening by using an empty needle cylinder, screwing a new empty tube at the right outer opening, connecting the left outer opening with a needle cylinder containing 30mL of complete culture medium, injecting the culture medium into the culture cylinder, and slowly pushing in the process to avoid generating bubbles;
(f) And closing the valve in the extrafibrous space and opening the valve in the intraductal pipe. The culture cylinders were loaded onto a double-ended pump, the flow rate was set to 25, and placed into an incubator to circulate for 48 hours, followed by circulating RoosterNourish in the system TM The complete medium of MSC-XF is replaced by fresh complete medium, and the seeding of cells can be started.
The present invention preferably performs the inoculation of cells after the pretreatment described above is completed, and the following should preferably be taken into account when preparing cells: a. shortening the time for harvesting the cells as much as possible so as to improve the survival rate of the cells; b. the survival rate of inoculated cells is more than 95 percent; c. the number of cells seeded is sufficient to cover 50% of the fiber surface area (1 x 10 cells seeded for C2011) 8 ) The method comprises the steps of carrying out a first treatment on the surface of the d. When the cells are inoculated, the supernatant fluid of the cells in the culture period is used for re-suspending and concentrating the cells, and the cells contain various growth factors, so that the growth of the cells is facilitated; concentrating the total volume of the cell fluid to 15mL, and sucking the cell fluid into a syringe. The inoculation according to the invention preferably comprises: 1) Material preparation: a syringe containing cell concentrate, 1 new syringe, 125mL complete medium, alcohol (for disinfection);
2) Sterilizing the culture medium bottle mouth, and replacing the circulating culture medium bottle with a new culture medium bottle;
3) Closing the inner pipe valve in the fiber, and opening the outer space valve after disinfection;
4) The right outer opening is screwed with an upper needle cylinder, and the left outer opening is screwed with a needle cylinder with cell concentrate; injecting cell concentrate from the left outer opening, and at the moment, flushing part of the cell concentrate and the culture medium into the right empty syringe by liquid pressure;
5) The left end and the right end are punched back and forth until the turbidity of the liquid in the needle cylinders at the two ends is consistent, which means that the cells are uniformly mixed;
6) The syringes at the two ends respectively retain equal volume of liquid, and the right outer valve is closed firstly;
7) Opening the right inner valve, and loosening the culture medium bottle mouth to balance the air pressure in the pipeline;
8) Injecting the liquid into the left outer needle cylinder, wherein the culture medium flows from the right inner tube to the liquid storage bottle;
9) Closing the left outer valve, opening the right outer valve, and injecting liquid to enable the culture medium to flow from the right inner pipe to the culture medium bottle;
10 Sampling from the liquid storage bottle to measure the glucose concentration and recording, screwing the bottle cap, closing the right outer valve, and opening the left inner valve;
11 Rotating the culture cylinder downwards for 180 degrees, standing for 30min, correcting the culture cylinder, and standing for 30min again to enable the cells to be uniformly attached to the surface of the fiber. Placing the culture cylinder on a pump for culture, and setting the initial flow rate to be 25;
12 Daily monitoring of glucose consumption in the culture medium to assess the growth status of the cells in the culture drum;
500mL of fresh RosterNourish was replaced when the glucose concentration consumed 60% TM MSC-XF complete medium, increasing the flow rate to 28;
500mL of fresh RosterNourish was replaced when the glucose concentration consumed 50% TM MSC-XF complete medium, increasing the flow rate to 30;
when the glucose concentration had consumed 50%, 500mL of fresh RosterCollet-EV Pro was replaced TM Complete medium, flow rate maintained 30; cell supernatants were collected every 2 days initially;
500mL of fresh RosterCollect-EV Pro was replaced every time the glucose concentration was depleted by 50% TM The flow rate of the complete medium was always kept at 30.
In the present invention, the inoculated cells are cultured in a RossterNourish TM The consumption of glucose is basically stable for 3-4 days when cultured under the condition of MSC-XF culture medium, and cells in a culture barrel are near to a full-load state. Subsequent exchange of the roosterCollect-EV Pro TM Under these conditions, the cells are in a vesicle-enhanced secretion, cell proliferation-inhibited state. Exchange of RoosterCollect-EV Pro for the first time TM The complete culture medium is D0, the culture is carried out for 34 days, about 12mL of cell supernatant is collected once every two days, the pretreatment of the sample is carried out before the fine purification of exosomes by 3500 Xg centrifugation for 60min at 4 ℃, and the supernatant sample after centrifugation is transferred into a new centrifuge tube for freezing and preserving at-80 ℃ for standby, so that repeated freezing and thawing are avoided.
The specific operation for collecting the supernatant according to the present invention preferably comprises: I. material preparation: 2 empty syringes;
II, closing the left internal valve, and loosening the culture medium bottle mouth to balance the air pressure in the pipeline;
III, opening a left outer valve to suck about 6mL of liquid;
IV, closing the left outer valve, opening the right outer valve, and sucking 6mL of liquid;
v, closing the right inner valve and the culture medium bottle mouth, and opening the left outer valve;
VI, punching back and forth for 5 times by using two needle cylinders to enable cells which are not attached to the surface of the fiber to fall off;
VII, concentrating the harvested cell supernatant on a syringe outside the left, and taking down the syringe;
and VIII, closing the valve of the external fiber space, opening the valve of the internal fiber pipe, and placing the culture cylinder back to the pump for culture.
The invention is based on the RooterNourish of the company RooterBio TM MSC-XF mesenchymal stem cell expansion medium and roosterCollect-EV Pro TM The exosome production culture medium establishes a mesenchymal stem cell exosome preparation process under a 3D culture environment in a vacuum fiber bioreactor of fiber cell System company.
The following examples are provided to illustrate in detail the preparation system and the preparation method of a high concentration mesenchymal stem cell exosome according to the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
The exosome production of umbilical cord mesenchymal stem cells isolated from umbilical cord tissue sources of 3 different infants is carried out on fiber cell C2011, so that the preparation of the exosome of the mesenchymal stem cells with high concentration and low impurity can be carried out stably for a long time under the process condition.
(1) Preparation of inoculated cells: at 1X 10 6 Inoculating P4 generation human umbilical cord mesenchymal stem cells (hMSC) into 15T-175 culture flasks, adding RossterNourish TM Cell expansion with MSC-XF complete Medium, replacement of fresh Medium after 2 washes with D-PBS on day 4, cell counting with cell collection on day 6, and cell quality testing including sterility testing and cell surfaceType test, while collecting 50mL of cell culture supernatant, using a syringe through a 0.22 μm filter membrane filtration into a new centrifuge tube, 37 ℃ storage for standby.
(2) Fiber Cell bioreactor pretreatment
Circulating PBS solution for 48 hours;
2. replacement to RoosterBasal TM -MSC-XF basal medium is circulated for 48h;
3. and then change into RossterNourish TM -MSC-XF complete medium circulation for 48h;
4. finally, replacing with fresh roosterNourish TM MSC-XF complete medium, cells can be inoculated.
The pattern of the fibercell exosome production system is shown in figure 1.
(3) Inoculating cells: 1. material preparation: a syringe containing a cell concentrate, 1 new syringe, 125mL of complete medium, alcohol (for sterilization).
2. And (3) sterilizing the culture medium bottle mouth, and replacing the circulating culture medium bottle with a new culture medium bottle.
3. And closing the intra-fiber pipe valve, and opening the extra-fiber space valve after disinfection.
4. The right outer opening is screwed with an upper needle cylinder, and the left outer opening is screwed with a needle cylinder with cell concentrate. Cell concentrate is injected from the left outer opening, and at this time, part of the cell concentrate and the culture medium are flushed into the right empty syringe by the liquid pressure.
5. The left end and the right end are punched back and forth until the turbidity of the liquid in the needle cylinders at the two ends is consistent, which means that the cells are uniformly mixed.
6. The syringes at the two ends respectively hold equal volumes of liquid, and the right outer valve is closed first.
7. And opening the right inner valve, and loosening the culture medium bottle mouth to balance the air pressure in the pipeline.
8. The liquid in the left outer syringe is injected, and the culture medium flows from the right inner tube to the liquid storage bottle.
9. The left outer valve is closed, the right outer valve is opened, and liquid is injected to enable the culture medium to flow from the right inner pipeline to the culture medium bottle.
10. The glucose concentration is measured by sampling from the liquid storage bottle and recorded, the bottle cap is screwed down, the right outer valve is closed, and the left inner valve is opened.
11. Rotating the culture cylinder downwards for 180 degrees, standing for 30min, correcting the culture cylinder, and standing for 30min again to enable the cells to be uniformly attached to the surface of the fiber. The culture cylinder was placed on a pump for cultivation, and the initial flow rate was set to 25.
12. The glucose consumption in the medium was measured daily to assess the growth status of the cells in the culture drum.
500mL of fresh RosterNourish was replaced when the glucose concentration consumed 60% TM MSC-XF complete medium, increasing the flow rate to 28.
500mL of fresh RosterNourish was replaced when the glucose concentration consumed 50% TM MSC-XF complete medium, increasing the flow rate to 30.
When the glucose concentration had consumed 50%, 500mL of fresh RosterCollet-EV Pro was replaced TM Complete medium, flow rate was maintained at 30. Cell supernatants were collected every 2 days.
500mL of fresh RosterCollect-EV Pro was replaced every time the glucose concentration was depleted by 50% TM The flow rate of the complete medium was always kept at 30.
(4) Harvesting the cell supernatant:
inoculated cells in the roosterNourish TM The glucose consumption is basically stable for 3-4 days when the MSC-XF is cultured under the complete culture medium condition, and the cells in the culture barrel are near to the full load state.
Subsequent exchange of the roosterCollect-EV Pro TM Complete medium, under which the cells are in a vesicle-enhanced secretion, cell proliferation-inhibited state.
Exchange of RoosterCollect-EV Pro for the first time TM The complete culture medium is D0, the culture is carried out for 34 days, about 12mL of cell supernatant is collected once every two days, the pretreatment of the sample is carried out before the fine purification of exosomes by 3500 Xg centrifugation for 60min at 4 ℃, and the supernatant sample after centrifugation is transferred into a new centrifuge tube for freezing and preserving at-80 ℃ for standby, so that repeated freezing and thawing are avoided.
Preparation of exosome to meet the requirement of umbilical cord-derived mesenchymal stem cells of different tissues
The parent cell and culture system determine the composition of the exosomes, while homogeneous cells of different tissue origin need to be used to verify the stability of the exosome preparation process to meet the inter-lot consistency requirements of GMP.
The invention selects 3 mesenchymal stem cells from different infant umbilical cord tissues as parent cells, and tests cell viability, phenotype and three-line differentiation capability before inoculation. The results are shown below: the mesenchymal stem cells hUC-MSC-0901, hUC-MSC-0214 and hUC-MSC-1103 from 3 different tissues have the activity rate of more than 95 percent (shown in figure 2a, figure 2b and figure 2 c), the mesenchymal stem cell surface markers CD73/90/105 is more than or equal to 95 percent, and CD45/34/11b/19/HLA-DR is less than or equal to 2 percent, and all have the lipid-forming, bone-forming and cartilage-forming three-line differentiation capacity (table 1) and meet the experimental requirements.
TABLE 1 three different umbilical cord tissue-derived mesenchymal stem cells P 5 Cell surface markers prior to generation inoculation and statistics of three-line differentiation capacity
(II) stable exosome particle size and yield during long term exosome harvest
Upon replacement with a RosterColllect-EV Pro TM When complete medium was used for exosome production, the present invention evaluates the extracellular vesicles from the hUC-MSC (0901, 0214, 1103) from different donors at the beginning of production (day 2), mid-production (day 14) and end of production (day 26). Sample nanoparticle concentrations and particle size distributions were measured at different time points using a nanoparticle tracking analysis instrument ZetaView.
Taking 0214 as an example, the result of NTA test particle concentration is shown in FIG. 3, and the concentrations of exosomes detected at Day2, day14, day26 are about 2.0E+10particles/mL,1.9E+10particles/mL, and 2.0E+10particles/mL, and the yields of exosomes are stable before, during and after production.
Also taking 0214 as an example, the particle size distribution results of NTA test particles are shown in FIG. 4, the particle size distribution range of exosomes detected during Day2, day14 and Day26 is between 105 and 120nm, the front-middle period accounts for more than 95%, the later period accounts for 90%, and the particle sizes of the rest particles are more than 200nm, and the exosomes are fused and related to long-term culture vesicles. The grain size of the exosomes is mainly distributed at 30-150 nm, and vesicles outside the range can be removed through fine purification, so that the grain size of the exosomes produced in the front, middle and later stages of production is stable.
The results of statistical analysis on the concentrations and particle sizes of the exosomes of the hUC-MSC-0901, 0214 and 1103 prove that the yield (Table 2) and the particle size distribution (Table 3) of the harvested exosomes of the parent cells from different umbilical cord tissues have good stability in the production process of the exosomes in 25 days.
TABLE 2 production of umbilical cord-derived mesenchymal Stem cell exosomes before, during and after harvesting
TABLE 3 particle size of umbilical cord-derived mesenchymal Stem cell exosomes before, during and after harvesting
(III) exosome composition is stable during long term exosome harvest
The invention uses a NanoView exosome analyzer to test exosome compositions at different times in the production stage, and can see that the exosome compositions of the UC-MSC-0214 production stage are nearly the same at the time points of D2, D14 and D26, and the compositions of the exosome in the CD63, CD81 and CD9 capturing channels (figure 5), and the particle sizes detected by the NanoView are not obviously different (figure 6), so that the stability of the exosome subgroup compositions in the front, middle and later stages of exosome production is proved.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
1. A preparation method of a high-concentration mesenchymal stem cell exosome, which is characterized by comprising the following steps: (1) Performing cell expansion on the mesenchymal stem cells by using a RoosterNourish ™ -MSC-XF complete medium, and respectively collecting cells and cell culture supernatant when the cells grow to 85% fusion degree;
(2) After the collected cells are resuspended by utilizing the cell culture fluid supernatant in the step (1), inoculating the cells into 3D culture equipment of the pretreated mesenchymal stem cells in a RoosterNourish ™ -MSC-XF complete culture medium environment, and replacing the culture medium according to the consumption of glucose in the culture medium; the 3D culture equipment of the mesenchymal stem cells comprises a hollow fiber bioreactor or full-automatic mesenchymal stem cell culture equipment with the same principle as the hollow fiber bioreactor;
the new roosternourise ™ -MSC-XF complete medium was exchanged for the first time when the glucose concentration was 60% depleted; the RooterNourish ™ -MSC-XF complete medium is formed by adding a RooterBooster ™ -MSC-XF additive into a RooterBasal ™ -MSC-XF basic medium;
a second replacement of the new roosternourise ™ -MSC-XF complete medium when the glucose concentration was depleted by 50%;
when the glucose concentration was depleted by 50%, a third change was made to the new RoosterCollect-EV Pro ™ complete medium;
after the third renewal, the culture was continued for 34 days again, and the cell supernatant was collected every two days, and the mesenchymal stem cell exosomes were extracted using the cell supernatant.
2. The process according to claim 1, wherein in step (1) the ratio of the catalyst to the catalyst is 1X 10 6 cell/mL, mesenchymal stem cells were inoculated into the RoosterNourish ™ -MSC-XF complete medium, cell expansion was performed, the cells were washed with D-PBS on day 4, the new RoosterNourish ™ -MSC-XF complete medium was replaced, and the cells and cell culture supernatants were collected, respectively, after the cells grew to 85% confluency.
3. The method of claim 1, wherein the pretreatment of step (2) comprises a first 48h cycle of the hollow fiber bioreactor with PBS solution, a second 48h cycle of the RossterBasal ™ -MSC-XF basal medium, and a third 48h cycle of the RossterNourish ™ -MSC-XF basal medium.
4. The method of claim 3, further comprising, after the third cycle, replacing the new RoosterNourish ™ -MSC-XF complete medium for cell seeding.
5. The method of claim 1, further comprising centrifuging 3500 Xg at 4℃for 60 minutes after collecting the cell supernatant, and collecting the supernatant.
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