CN202187007U - Umbilical cord MSCs digitization automatic production system - Google Patents
Umbilical cord MSCs digitization automatic production system Download PDFInfo
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Abstract
The utility model relates to an umbilical cord MSCs (Mesenchymal Stem Cell) digitization automatic production system, which is built by combing culture flasks taken as cell growth carriers, a mechanical assembly line and a digitization full-automatic biological reactor. The culture flasks comprise three culture flasks placed on the mechanical assembly line and circumscribed with the biological reactor. The mechanical assembly line comprises a mechanical transmission device and an electronic control device, wherein the electronic control device controls the mechanical transmission device according to processing data of the biological reactor, so as to enable the culture flasks to complete actions such as oscillation, turnover, 90 DEG tilt and the like in the assembly line. The biological reactor comprises a control system, a detection device, an air supply device, a liquid adding device and a liquid removing device, monitors key parameters in a cell culture process via the detection device, analyzes and processes the key parameters, and then cooperates with the mechanical assembly line to automatically complete operations such as air supply, liquid adding, liquid removing and liquid collecting, so as to realize the entire cell culture process. Therefore, by adopting the above technical scheme, umbilical cord organization blocks can increase MSCs to 109 in the automatic production system.
Description
Technical field
The utility model belongs to cell cultures and cell engineering field, relates to umbilical cord mesenchymal stem cells digitizing Automatic Production System.
Background technology
Traditional cell cultures mostly is manual operation, all respects of producing is had many restrictions, such as peopleware, clean working spaces rank, operational administrative rules and regulations etc.Although possessed the production management of severization and perfect quality control, the influence that many human factors are produced stem cell also is that inevitably the quality of cell and stability still can not guarantee.At present, bio-reactor is at home and abroad extensively carried out such as the cell scale prodn field of production of vaccine, and it can improve the production efficiency of cell and associated products effectively and reduce the risk of cells produce.Yet, the still immature mescenchymal stem cell that is applied to of bio-reactor, major cause is: 1. these technical qualification are higher, need select suitable carriers for use so that cell attaches and cultivation under three-dimensional weightless environment; 2. three-dimensional weightless cell culture environment has many differences with traditional static cultivation, and the cell quality of therefore producing can not know that still also there is certain risk in this clinical application to mescenchymal stem cell; 3. curative effect and the security in order to guarantee the mescenchymal stem cell clinical application in the practice do not allow long-time amplification, need not too many cell yet, and this has also limited the application of the bio-reactor of traditional scale operation cell.
The utility model content
Waste time and energy in order to solve in the prior art artificial culture cell, cell cultures poor stability, deficiency such as cost is high, efficient is low; The utility model provides a kind of umbilical cord mesenchymal stem cells digitizing Automatic Production System; This system applies is cultivated in robotization, the flow line of cell, has reduced the manual operation influence factor, guarantees the quality and the stability of cell cultures; Practice thrift the cell cultures cost, improve cell cultures efficient.
The technical scheme that the utility model adopts is: a kind of umbilical cord mesenchymal stem cells digitizing Automatic Production System; Comprise culturing bottle, mechanical flow line, bio-reactor; Bio-reactor comprises system, means of detection, air feeder, liquid-adding device, removes liquid device, numeral shows touch-screen; Its technical characterictic is that said culturing bottle comprises at least three culturing bottles; At least three culturing bottle average levels are positioned on the mechanical flow line, and air feeder, the means of detection of bio-reactor all is set on the bottle cap of three culturing bottles at least; First culturing bottle of at least three culturing bottles is the monolayer culture bottle of cultivation of primary cells; The bottle end of first culturing bottle, is provided with four interfaces and is respectively liquid feeding interface A1, tissue block interpolation interface A2, removes liquid interface A3 and collect interface A4; Liquid feeding interface A1 with go liquid interface A3 all to be connected bio-reactor; The effect of liquid feeding interface A1 is from bio-reactor, to add the required related solution of cell cultures such as nutrient solution, Digestive system; The effect that tissue block is added interface A2 is to add umbilical cord China Tong Shi glue tissue block; I.e. sealing after the interpolation, go liquid interface A3 be with solution such as the nutrient solution of culturing cell, Digestive system from then on interface shift to bio-reactor, collect interface A4 and be used for collecting primary cell; Said second culturing bottle is the passage cell culturing bottle; The bottle end of second culturing bottle, is provided with four interfaces and is respectively liquid feeding interface B1, cell interpolation interface B2, removes liquid interface B3 and collect interface B4; Liquid feeding interface B1 with go liquid interface B3 all to be connected bio-reactor; The effect of liquid feeding interface B1 is from bio-reactor, to add the required related solution of cell cultures such as nutrient solution, Digestive system; Cell adds interface B2 and connects first culturing bottle and collect interface A4 and receive primary cell, B3 be with solution such as the nutrient solution of culturing cell, Digestive system from then on interface shift to bio-reactor, B4 is used for collecting cell; Said the 3rd culturing bottle is the passage cell culturing bottle; The bottle bottom of the 3rd culturing bottle is provided with four interfaces and is respectively liquid feeding interface C1, cell interpolation interface C2, removes liquid interface C3 and collect interface C4; Liquid feeding interface C1 with go liquid interface C3 all to be connected bio-reactor; Cell adds interface C2 and connects second culturing bottle collection interface B4; Liquid feeding interface C1 effect is from bio-reactor, to add the required related solution of cell cultures such as nutrient solution, Digestive system, and cell adds interface C2 and connects second culturing bottle and collect interface B4 and receive the second culturing bottle cultured cells, go liquid interface C3 be with solution such as the nutrient solution of culturing cell, Digestive system from then on interface shift to bio-reactor; Collect interface C4 and in cell cultivation process, seal, external cell harvesting container when cultivating the cell harvesting of accomplishing.
Further, the liquid-adding device in the said liquid feeding interface A1, B1, the external bio-reactor of C1, and liquid feeding interface A1, B1, the last 0.22 μ m strainer that is equipped with of C1 prevent that liquid feeding from polluting; The said liquid device that goes that goes in liquid interface A3, B3, the external bio-reactor of C3, and go liquid interface A3 to be provided with the screen cloth that pore size is 2mm, tissue block shifts out when preventing liquid; Said collection interface A4, B4, last 200 eye mesh screens that are equipped with of C4 prevent not to be digested to single celled cell mass and shift out.
Further, several interfaces at the bottom of said first, second and third culturing bottle bottle all are arranged at top or the tip position at the bottom of the culturing bottle bottle, and several interfaces all are on the same horizontal plane.When the culturing bottle flip horizontal is inverted; Several interfaces all are in the below; Make things convenient for liquid successfully to flow out, when the controlled upset of culturing bottle is the vertical state of 90 degree, collects interface and all be positioned at below of culturing bottle by the corresponding interface; Liquid or cell etc. can be from the smooth and easy outflows of culturing bottle, and the convenient passage of execution fast changes the relating operation of liquid.
Further; Be provided with multi-layer cellular in said second and third culturing bottle and cultivate the plane; Said every layer of cultivation both sides, plane all join with the Tissue Culture Flask both sides; Cultivate other both sides, plane for every layer and separate with bottle cap with the bottle end of Tissue Culture Flask and be provided with and exceed the border of cultivating plane 4mm and surround the pond shape, it is the parallel setting that do not contact each other that the liquid below the horizontal splendid attire 4mm height, multi-layer cellular are cultivated the plane.
Again further, can set up one or more passage cell culturing bottles identical between said second culturing bottle and the 3rd culturing bottle with the second culturing bottle structure.
The described mechanical flow line of the utility model comprises electronic controls and at least three group mechanical drives that are connected with electronic controls respectively; Said each mechanical drive comprises hanging up, the movement arm that drives hanging up and the motor of drive actions arm respectively; Electronic controls is connected with the system of bio-reactor; After handling, the data analysis that the means of detection of bio-reactor is surveyed is transferred to the electronic controls of mechanical flow line; Electronic controls is controlled at least three group mechanical drives respectively; Make the culturing bottle that is arranged at respectively on the mechanical drive controlledly be vibration, tilt, flip upside down or the vertical state of 90 degree, cooperate cell cultures to accomplish relevant action.Also be provided with the heating unit that is connected with electronic controls on the hanging up of each mechanical drive, realize that cell cultivation process needs the heating of culturing bottle.
The described bio-reactor of the utility model comprises system, means of detection, air feeder, liquid-adding device, removes liquid device, numeral shows touch-screen; Said means of detection, air feeder, liquid-adding device, go liquid device, numeral to show that touch-screen all is connected with system; Said means of detection comprises some probes such as carbonic acid gas probe, temp probe, PH probe; The every probe of means of detection and the air-supply duct of air feeder all are arranged on the bottle cap of every culturing bottle; Every probe stretches in the Tissue Culture Flask desired parameters in the detection of cells culturing process by the bottleneck of each culturing bottle, and detectable signal is sent to system; Air feeder is that cell growth provides required gas and keeps between each culturing bottle air pressure constant, the air demand of air feeder, the liquid volume added of liquid-adding device and go the liquid measure of liquid device to control by system; Liquid feeding interface C1 at the bottom of liquid feeding interface B1 at the bottom of liquid feeding interface A1 on said liquid-adding device connects at the bottom of the first culturing bottle bottle, the second culturing bottle bottle and the 3rd culturing bottle bottle; Go liquid device on connecting at the bottom of the first culturing bottle bottle go at the bottom of liquid interface A3, the second culturing bottle bottle on go at the bottom of liquid interface B3 and the 3rd culturing bottle bottle on remove liquid interface C3, the transfer of liquid is used when changing liquid and go down to posterity for cell; The system of bio-reactor with detectable signal analyzing and processing of each probe of means of detection after to liquid-adding device, go liquid device, air feeder to send liquid feeding, remove liquid, tonifying Qi information, and cooperate to the electronic controls of the mechanical flow line of control data information transfer and accomplish the mechanical action that goes down to posterity, changes cell cultivation process such as liquid.Be provided with medical saline in the said liquid-adding device, need not the neutral digestive ferment, the clinical grade cell culture fluid.Said numeral shows the monitoring data that touch-screen is conveniently checked cell cultivation process.
The Automatic Production System of the utility model gets into the cell cultures state, and the means of detection of bio-reactor is surveyed the parameters of culturing process; CO in the air feeder control cell growth process of bio-reactor
2Concentration remain at 5%, and keep between each culturing bottle air pressure constant; Nutritive substance consumption is too much in nutrient solution; It is following that the liquid step is changed in bio-reactor and mechanical flow line co-operating: the culturing bottle controlled oscillation flipped upside down after 10 seconds; Nutrient solution is by going liquid interface to remove the liquid device that goes to bio-reactor; The controlled upset of culturing bottle is just put, and from the liquid-adding device of bio-reactor, in culturing bottle separately, replenishes new nutrient solution through liquid feeding interface, three culturing bottles controlled on mechanical flow line single movement; The machinery flow line keeps 37 ℃ of the cell cultures temperature of each culturing bottle constant, and regular small vibration, guarantees the homogeneity of cell growth.The system of bio-reactor is through detection each item key parameter variation tendency; Analyze, judge cell growth condition in the culturing bottle; And the amount of the required various liquid of the time and the cell dissociation that calculate passage; Bio-reactor and the mechanical flow line co-operating step that goes down to posterity is following: the culturing bottle controlled oscillation flipped upside down after 10 seconds; Nutrient solution is by going liquid interface to remove the liquid device that goes to bio-reactor, and the controlled upset of culturing bottle is just put, and in culturing bottle separately, replenishes saline water from the liquid-adding device of bio-reactor through liquid feeding interface; The machinery flow line vibrates and washs for 10 seconds; Culturing bottle is controlled to flip upside down; Saline water removes the liquid device that goes to bio-reactor by the liquid interface that goes of culturing bottle; The controlled upset of culturing bottle is just put, and in culturing bottle, replenishes the required enzyme of cell dissociation through liquid feeding interface again from the liquid-adding device of bio-reactor; 37 ℃ of digestion of machinery flow line shaking culture bottle 5 minutes; The controlled upset of culturing bottle is 90 degree vertically; Cell suspension is interfaced to next culturing bottle through collection and cultivates or directly be moved to the cell harvesting container; The controlled upset of culturing bottle is just put, and the liquid-adding device from bio-reactor replenishes a small amount of nutrient solution through liquid feeding interface in culturing bottle again, and the culturing bottle controlled oscillation overturns after 10 seconds again and is 90 degree vertically; Through the collection interface of culturing bottle with residual cell transfer to next culturing bottle or be collected in the cell harvesting container, three culturing bottles controlled on mechanical flow line single movement.Operation is just being put in upset, and is even for guaranteeing each cell cultures layer Liquid Distribution, by just putting rapidly behind mechanical flow line control the second or the 3rd culturing bottle rollover 90 degree; The operation that flips upside down for solution such as the nutrient solution that guarantees second, third each culture layer of culturing bottle, Digestive system all can remove fully, slowly is inverted after stopping for 5 seconds by mechanical flow line control the second or the 3rd culturing bottle rollover 90 degree backs.
After adopting above technical scheme, the beneficial effect of the utility model is:
Actions such as the mechanical flow line that the utility model adopts cooperates bio-reactor to accomplish cell cultures automatically, changes liquid, goes down to posterity, the vibration of collecting processes, upset, inclination 90 degree, the cultivation that has realized cell is by the transformation of manual work to robotization; In culturing process, can slightly rock, guarantee cell former be commissioned to train foster evenly, the cell mass discrepancy is few; Cell cultures adopts the multi-layer cellular culturing bottle to leave standstill cultivation, can guarantee that cultured cells quality and artificial conventional cultured cells are as good as; Multilayer is cultivated the plane stack and has been saved the space in the culturing bottle, has reduced the needs of bio-reactor being surveyed the probe number, has also saved other auxiliary facility, and system's use cost is low; Adopt the full-automatic bio-reactor pair cell culturing process visualization digital monitoring of digital display; Can accurate calculation go down to posterity and change liquid time, change the consumption of various liquid in liquid measure, the digestive process, realize the accurate control of cells produce whole process, avoid the manual operation factor affecting; Reduce the waste of cultivated material; The cell steady quality of turning out satisfies the digitizing of the large scale culturing of cell and monitors high efficiency in real time; By mechanical flow line and the collaborative whole process of accomplishing the cell automatic production of bio-reactor, adopt totally-enclosed mode to operate, reduced the possibility of polluting, reduced the expense of clean room and pertinent instruments consumptive material.
The digitizing Automatic Production System of the utility model is cultivated through robotization, the flow line of cell cultures whole process; Reduce the manual operation influence factor, improved the training quality of cell greatly, improved the stability of cell; Practice thrift the cell cultures cost, cell cultures efficient significantly improves.
Description of drawings
Fig. 1 is the utility model structural representation.
Among Fig. 1: bio-reactor 1, system 11, liquid-adding device 12 removes liquid device 13, air feeder 14, means of detection 15; Machinery flow line 2, electronic controls 21, motor 22, movement arm 23, hanging up 24,25,26; First culturing bottle 3, second culturing bottle, 4, the three culturing bottles 5.
Embodiment
Below in conjunction with accompanying drawing the utility model is done further explain.
A kind of umbilical cord mesenchymal stem cells digitizing Automatic Production System in shown in Figure 1 comprises culturing bottle, mechanical flow line 2, bio-reactor 1.Described mechanical flow line 2 comprises electronic controls 21 and at least three group mechanical drives that are connected with electronic controls respectively; Said each mechanical drive comprises hanging up 24 respectively, drives the movement arm 23 of hanging up motion and the motor 22 of drive actions arm work; Motor connects electronic controls; Electronic controls is connected with the system 11 of bio-reactor; Place a culturing bottle on every hanging up, also be provided with the heating unit that is connected with electronic controls on each hanging up, realize that cell cultivation process needs the heating of culturing bottle.
The described bio-reactor 1 of the utility model comprises system 11, means of detection 15, air feeder 14, liquid-adding device 12, goes liquid device 13 and numeral to show touch-screen; Said means of detection 15, air feeder 14, liquid-adding device 12, go liquid device 13, numeral to show that touch-screen all is connected with system 11; Said means of detection 15 comprises some probes such as carbonic acid gas probe, temp probe, PH probe; The every probe of means of detection and the air-supply duct of air feeder all are arranged on the bottle cap of every culturing bottle; Every probe stretches in the Tissue Culture Flask desired parameters in the detection of cells culturing process by the bottleneck of each culturing bottle, and detectable signal is sent to system; Air feeder 14 provides required gas for cell growth and keeps between each culturing bottle air pressure constant, the liquid volume added of the air demand of air feeder, liquid-adding device 12 and remove liquid device 13 go liquid measure by system 11 controls.Send tonifying Qi, liquid feeding, remove liquid after bio-reactor 1 detectable signal analyzing and processing each probe of means of detection, acquisition of information; And make the culturing bottle on the hanging up accomplish the vibration of cell cultivation process, tilt, turn over and mechanical action such as turn 90 degrees to the electronic controls 21 of mechanical flow line 2,23 motions of electronic controls control action arm the data information transfer; Be provided with medical saline in the liquid-adding device 12, need not the neutral digestive ferment, the clinical grade cell culture fluid.
Said culturing bottle comprises at least three culturing bottles 3,4,5; The bottle cap of at least three culturing bottles and bottle coexist at an end and to be positioned on the hanging up 24,25,26 of mechanical flow line 2 on the horizontal plane, and air feeder 14, the means of detection 15 of bio-reactor all is set on the bottle cap of three culturing bottles at least; First culturing bottle 3 of at least three culturing bottles is the monolayer culture bottle of primary cell, and second and third culturing bottle 4,5 is provided with multi-layer cellular in being and cultivates the planar culturing bottle that goes down to posterity; The bottle end of first culturing bottle 3, is provided with four interfaces and is respectively liquid feeding interface A1, tissue block interpolation interface A2, removes liquid interface A3 and collect interface A4; The bottle end of second culturing bottle 4, is provided with four interfaces and is respectively liquid feeding interface B1, cell interpolation interface B2, removes liquid interface B3 and collect interface B4; The bottle bottom of the 3rd culturing bottle 5 is provided with four interfaces and is respectively liquid feeding interface C1, cell interpolation interface C2, removes liquid interface C3 and collect interface C4; The liquid feeding interface C1 of the liquid feeding interface B1 of the liquid feeding interface A1 of first culturing bottle 3, second culturing bottle 4, the 3rd culturing bottle 5 all connects the liquid-adding device 12 of bio-reactor 1; Effect is from the liquid-adding device of bio-reactor, to add the required related solution of cell cultures such as nutrient solution, Digestive system through liquid feeding interface; And liquid feeding interface A1, B1, the last 0.22 μ m strainer that is equipped with of C1 prevent that liquid feeding from polluting; First culturing bottle 3 go liquid interface A3, second culturing bottle 4 go liquid interface B3, the 3rd culturing bottle 5 go that liquid interface C3 all connects bio-reactor 1 remove liquid device 13; Effect is that solution such as the nutrient solution of culturing cell, Digestive system are shifted to the liquid device that goes of bio-reactor from removing liquid interface; And the liquid interface A3 that goes of first culturing bottle 3 is provided with the screen cloth that pore size is 2mm, and tissue block shifts out when preventing that first culturing bottle from removing liquid; It is the round interface of diameter 1cm that the tissue block of first culturing bottle 3 is added interface A2; Its effect is to add umbilical cord China Tong Shi glue tissue block; I.e. sealing after the interpolation; The collection interface A4 of first culturing bottle 3 is the cell interpolation interface B2 that the primary cell of collecting delivered to second culturing bottle 4; The cell that the collection interface B4 of second culturing bottle 4 delivers to the 3rd culturing bottle 5 with the passage cell of collecting adds interface C2, and in cell harvesting to the cell harvesting container that the collection interface C4 through the 3rd culturing bottle 5 will finally turn out, the 3rd culturing bottle 5 is collected interface C4 and in cell cultivation process, sealed; And collect interface A4, B4, last 200 eye mesh screens that are equipped with of C4, prevent not to be digested to single celled cell mass and shift out.Second culturing bottle 4 can be set up one or more passage cell culturing bottles identical with the second culturing bottle structure with 5 on the 3rd culturing bottle.
Further, several interfaces at the bottom of 3,4,5 bottles on said first, second and third culturing bottle all are arranged at top or the tip position at the bottom of the culturing bottle bottle, and several interfaces all are on the same horizontal plane.When the culturing bottle flip horizontal is inverted; Several interfaces all are in the below; Make things convenient for liquid successfully to flow out, when the controlled upset of culturing bottle is the vertical state of 90 degree, collects interface and all be positioned at below of culturing bottle by the corresponding interface; Liquid or cell etc. can be from the smooth and easy outflows of culturing bottle, and the convenient passage of execution fast changes the relating operation of liquid.
Be equipped with multi-layer cellular cultivation plane in said second and third culturing bottle 4,5 or in the culturing bottle of setting up between the second and the 3rd culturing bottle; Cultivating the planar both sides for every layer all joins with the Tissue Culture Flask both sides; Cultivating other both sides, plane for every layer separates with bottle cap with the bottle end of Tissue Culture Flask and is provided with the border that exceeds cultivation plane 4mm; Can enclose the pond shape with the culturing bottle both sides, it is the parallel setting that do not contact each other that the liquid below the horizontal splendid attire 4mm height, multi-layer cellular are cultivated the plane.During liquid feeding; Even for guaranteeing each cell cultures layer Liquid Distribution of second, third culturing bottle, by just putting rapidly behind mechanical flow line 2 control culturing bottle rollovers 90 degree, when culturing bottle rollover 90 degree states; Liquid comes together in bottle side lower part and the bottle end and bottle cap place; When the culturing bottle instant reverse was just being put, liquid can be evenly distributed to each layer cultivation plane immediately in the culturing bottle, has made full use of the useful space of culturing bottle; When removing liquid, culturing bottle rollover 90 degree backs stopped for 5 seconds, and liquid comes together in bottle side lower part and the bottle end and bottle cap place; Slowly be inverted; Liquid promptly is pooled to below of culturing bottle fully, and liquid or cell etc. can be from the smooth and easy outflows of culturing bottle, conveniently carries out the relating operation that passage changes liquid fast.
The step that the utility model is applied to cell cultures is following:
(1) umbilical cord with aseptic collection is cut into 1mm
3Tissue block; Tissue block is resuspended after the tissue block of first culturing bottle is added interface A2 adding through nutrient solution; Close tissue block then and add interface A2, system gets into the cell cultures state, and the means of detection of bio-reactor is surveyed the parameters of culturing process;
(2) bio-reactor control air feeder makes CO in the cell growth process
2Concentration remain at 5%, and keep culturing bottle air pressure constant; Nutritive substance consumption is too much in nutrient solution; It is following that liquid is changed in bio-reactor and mechanical flow line co-operating: first culturing bottle be former generation tissue block culturing bottle controlled oscillation flip upside down after 10 seconds; Nutrient solution is by going liquid interface A3 to get rid of; The controlled upset of first culturing bottle is just put, and replenishes new nutrient solution through liquid feeding interface A1;
(3) mechanical flow line keeps in first culturing bottle 37 ℃ of cell cultures temperature constant, and regular small vibration, guarantees the adherent homogeneous of primary cell;
(4) means of detection of bio-reactor is analyzed, is judged the cell growth condition in first culturing bottle, and calculate the time of passage, the amount of the various liquid that cell dissociation is required through surveying each item key parameter variation tendency;
During (5) to passage, first culturing bottle is controlled to flip upside down, and nutrient solution is by going liquid interface A3 to get rid of; The controlled upset of first culturing bottle is just put, and replenishes saline water through liquid feeding interface A1, and mechanical flow line vibration makes first culturing bottle wash for 10 seconds; First culturing bottle is controlled to flip upside down, and saline water is by going liquid interface A3 to get rid of, and the controlled upset of first culturing bottle is just put; Replenish the required enzyme of cell dissociation through liquid feeding interface A1 again, 37 ℃ of digestion of mechanical flow line shaking culture bottle 5 minutes, the controlled upset of first culturing bottle is 90 degree vertically; Make cell suspension through collecting the cell interpolation interface B2 that interface A4 is transferred to second culturing bottle; The controlled upset of first culturing bottle is just put, and replenishes a small amount of nutrient solution through liquid feeding interface A1 again, 10 seconds of mechanical flow line shaking culture bottle; The controlled upset of first culturing bottle is 90 degree vertically, makes the residual cell suspension through collecting the cell interpolation interface B2 that interface A4 is transferred to second culturing bottle;
(6) first culturing bottle is controlled resets, and replenishes nutrient solution through liquid feeding interface A1, and bottle Central Plains continues to cultivate for tissue block;
Just putting to guarantee each cell cultures layer Liquid Distribution evenly behind the controlled rollover of (7) second culturing bottles 90 degree rapidly, leaving standstill to cultivate and made cell attachment in 12 hours, slowly be inverted after the controlled rollover of second culturing bottle 90 degree backs stopped for 5 seconds; Nutrient solution is got rid of through removing liquid interface B3; The controlled upset of second culturing bottle is just put, and saline water adds through liquid feeding interface B1, just puts rapidly behind controlled rollover 90 degree of second culturing bottle; The vibration of machinery flow line washed for 10 seconds; Slowly be inverted after the controlled rollover of second culturing bottle 90 degree backs stopped for 5 seconds, saline water is by going liquid interface B3 to get rid of, and the controlled upset of second culturing bottle is just put; Add new nutrient solution through liquid feeding interface B1, cultivation is left standstill in the controlled rollover rapidly positive postpone in 90 degree backs of second culturing bottle; Cell growth condition in second culturing bottle is analyzed, judged to means of detection through surveying each item key parameter variation tendency, and calculate the amount of the required various liquid of time and the cell dissociation of passage;
During (8) to passage, slowly be inverted after the controlled rollover of second culturing bottle 90 degree backs stopped for 5 seconds, nutrient solution is by going liquid interface B3 to get rid of; The controlled upset of culturing bottle is just put, and replenishes saline water through liquid feeding interface B1, just puts rapidly behind controlled rollover 90 degree of second culturing bottle; Machinery flow line shaking culture bottle washed for 10 seconds, slowly was inverted after the controlled rollover of culturing bottle 90 degree backs stopped for 5 seconds, and saline water is by going liquid interface B3 to get rid of; The controlled upset of culturing bottle is just put, and replenishes the required enzyme of cell dissociation through liquid feeding interface B1 again, just puts rapidly behind controlled rollover 90 degree of second culturing bottle; 37 ℃ of digestion of machinery flow line shaking culture bottle 5 minutes, the controlled upset of culturing bottle is the vertical state of 90 degree, makes cell suspension through collecting the cell interpolation interface C2 that interface B4 is transferred to the 3rd culturing bottle; The controlled upset of second culturing bottle is just put; Replenish a small amount of nutrient solution through liquid feeding interface B1 again, just put rapidly behind controlled rollover 90 degree of second culturing bottle, 10 seconds of mechanical flow line shaking culture bottle; The controlled upset of second culturing bottle is the vertical state of 90 degree, makes the residual cell suspension through collecting the cell interpolation interface C2 that interface B4 is transferred to the 3rd culturing bottle;
(9) second culturing bottle is controlled resets, and waits for that the cell that from first culturing bottle, digests moves into cultivation once more;
Just putting to guarantee each cell cultures layer Liquid Distribution evenly behind the controlled rollover of (10) the 3rd culturing bottles 90 degree rapidly, leaving standstill to cultivate and made cell attachment in 12 hours, slowly be inverted after the controlled rollover of the 3rd culturing bottle 90 degree backs stopped for 5 seconds; Nutrient solution is got rid of through removing liquid interface C3; The controlled upset of the 3rd culturing bottle is just put, and saline water adds through liquid feeding interface C1, just puts rapidly behind controlled rollover 90 degree of the 3rd culturing bottle; The vibration of machinery flow line washed for 10 seconds; Slowly be inverted after the controlled rollover of the 3rd culturing bottle 90 degree backs stopped for 5 seconds, saline water is by going liquid interface C3 to get rid of, and the controlled upset of the 3rd culturing bottle is just put; Add new nutrient solution through liquid feeding interface C1, cultivation is left standstill in the controlled rollover rapidly positive postpone in 90 degree backs of the 3rd culturing bottle; Cell growth condition in the 3rd culturing bottle is analyzed, judged to means of detection through surveying each item key parameter variation tendency, and calculate the amount of the required various liquid of time and the cell dissociation of passage;
(11) during passage, slowly be inverted after the controlled rollover of the 3rd culturing bottle 90 degree backs stopped for 5 seconds, nutrient solution is by going liquid interface C3 to get rid of; The controlled upset of culturing bottle is just put, and replenishes saline water through liquid feeding interface C1, just puts rapidly behind controlled rollover 90 degree of the 3rd culturing bottle; Machinery flow line shaking culture bottle washed for 10 seconds, slowly was inverted after the controlled rollover of culturing bottle 90 degree backs stopped for 5 seconds, and saline water is by going liquid interface C3 to get rid of; The controlled upset of culturing bottle is just put, and replenishes the required enzyme of cell dissociation through liquid feeding interface C1 again, just puts rapidly behind controlled rollover 90 degree of the 3rd culturing bottle; 37 ℃ of digestion of machinery flow line shaking culture bottle 5 minutes, the controlled upset of culturing bottle is the vertical state of 90 degree, and cell suspension is transferred in the cell harvesting container through collecting interface C4; The controlled upset of the 3rd culturing bottle is just put; Replenish a small amount of nutrient solution through liquid feeding interface C1 again, just put rapidly behind controlled rollover 90 degree of the 3rd culturing bottle, 10 seconds of mechanical flow line shaking culture bottle; Slowly be inverted after the controlled rollover of the 3rd culturing bottle 90 degree backs stopped for 5 seconds, the residual cell suspension is transferred in the cell harvesting container through collecting interface C4;
(12) cell that from first culturing bottle, grows, after repetition above-mentioned steps (2)-(11) are operated 3 times, the EO that the umbilical cord mesenchymal stem cells in this source is cultivated;
(13) cell of collecting in the cell harvesting container can obtain the umbilical cord mesenchymal stem cells of clinical usefulness through simple washing.
Claims (4)
1. umbilical cord mesenchymal stem cells digitizing Automatic Production System; Comprise culturing bottle, mechanical flow line (2), bio-reactor (1); Bio-reactor comprises system (11), means of detection (15), air feeder (14), liquid-adding device (12), goes liquid device (13) and numeral to show touch-screen; It is characterized in that: said culturing bottle comprises at least three culturing bottles (3,4,5); At least three culturing bottles all are arranged on the mechanical flow line (2), and the air feeder (14) and the means of detection (15) of bio-reactor all is set on the bottle cap of three culturing bottles at least; First culturing bottle (3) of at least three culturing bottles is the primary cell culture bottle; The bottle of first culturing bottle is provided with that four interfaces are respectively liquid feeding interface A1, tissue block is added interface A2, removes liquid interface A3 and collected interface A4 an end, liquid feeding interface A1 with go liquid interface A3 all to be connected bio-reactor; Said second culturing bottle (4) is the passage cell culturing bottle; The bottle end of second culturing bottle, is provided with four interfaces and is respectively liquid feeding interface B1, cell interpolation interface B2, removes liquid interface B3 and collect interface B4; Cell adds interface B2 and connects first culturing bottle and collect interface A4, liquid feeding interface B1 with go liquid interface B3 all to be connected bio-reactor; Said the 3rd culturing bottle (5) is the passage cell culturing bottle; The bottle bottom of the 3rd culturing bottle is provided with four interfaces and is respectively liquid feeding interface C1, cell interpolation interface C2, removes liquid interface C3 and collect interface C4; Cell adds interface C2 and connects second culturing bottle and collect interface B4, liquid feeding interface C1 with go liquid interface C3 all to be connected bio-reactor.
2. umbilical cord mesenchymal stem cells digitizing Automatic Production System according to claim 1 is characterized in that: the liquid-adding device in the said liquid feeding interface A1, B1, the external bio-reactor of C1, and liquid feeding interface A1, B1, C1 are provided with strainer; The said liquid device that goes that goes in liquid interface A3, B3, the external bio-reactor of C3, and go liquid interface A3 to be provided with screen cloth; Said collection interface A4, B4, C4 are provided with screen cloth.
3. umbilical cord mesenchymal stem cells digitizing Automatic Production System according to claim 1; It is characterized in that: said second and third culturing bottle (4; 5) be provided with multi-layer cellular in and cultivate the plane; Cultivate the both sides, plane and all join with the Tissue Culture Flask both sides for said every layer, cultivate other both sides, plane for every layer and separate with bottle cap and be provided with the border that exceeds cultivation plane 4mm with the bottle end of Tissue Culture Flask and surround the pond shape, multi-layer cellular cultivation plane is the parallel setting that do not contact each other.
4. umbilical cord mesenchymal stem cells digitizing Automatic Production System according to claim 1 is characterized in that: can set up one or more passage cell culturing bottles identical with the second culturing bottle structure between said second culturing bottle (4) and the 3rd culturing bottle (5).
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| CN 201120222170 CN202187007U (en) | 2011-06-28 | 2011-06-28 | Umbilical cord MSCs digitization automatic production system |
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| CN 201120222170 CN202187007U (en) | 2011-06-28 | 2011-06-28 | Umbilical cord MSCs digitization automatic production system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106244455A (en) * | 2016-08-29 | 2016-12-21 | 宁波键生物科技有限公司 | Fully-automatic intelligent cell culture system and control method thereof |
| CN106367343A (en) * | 2016-08-29 | 2017-02-01 | 宁波键生物科技有限公司 | Full-automatic intelligent cell culture device and control method thereof |
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2011
- 2011-06-28 CN CN 201120222170 patent/CN202187007U/en not_active Expired - Lifetime
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| CN106244455A (en) * | 2016-08-29 | 2016-12-21 | 宁波键生物科技有限公司 | Fully-automatic intelligent cell culture system and control method thereof |
| CN106367343A (en) * | 2016-08-29 | 2017-02-01 | 宁波键生物科技有限公司 | Full-automatic intelligent cell culture device and control method thereof |
| CN106367343B (en) * | 2016-08-29 | 2018-09-21 | 杭州键一生物科技有限公司 | A kind of fully-automatic intelligent cell culture apparatus and its control method |
| CN106244455B (en) * | 2016-08-29 | 2018-09-21 | 杭州键一生物科技有限公司 | Fully-automatic intelligent cell culture apparatus and its control method |
| CN109863240A (en) * | 2016-10-31 | 2019-06-07 | 通用电气公司 | Biological respinse device assembly |
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| CN111601878A (en) * | 2018-01-24 | 2020-08-28 | 横河电机株式会社 | Culture additive diffusion mechanism, culture container, culture system, and cell production method |
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