CN113632786A - Multi-chamber cryopreservation method for umbilical cord blood hematopoietic stem cells - Google Patents
Multi-chamber cryopreservation method for umbilical cord blood hematopoietic stem cells Download PDFInfo
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- CN113632786A CN113632786A CN202110952282.5A CN202110952282A CN113632786A CN 113632786 A CN113632786 A CN 113632786A CN 202110952282 A CN202110952282 A CN 202110952282A CN 113632786 A CN113632786 A CN 113632786A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
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Abstract
The invention discloses a multi-chamber cryopreservation method for umbilical cord blood hematopoietic stem cells, which comprises the following steps: primary transfer, extraction, precooling, mixing uniformly and reserving samples, exhausting, secondary transfer and reserving samples for storage: step A, primary transfer: transferring the cord blood hematopoietic stem cells obtained by centrifugation into a mixing bag structure, mixing uniformly, reserving a sample and weighing; and B, extraction: and (3) extracting the cryoprotectant according to the number of the information terminal by using an injector, wherein the cryoprotectant is prepared from dextran and dimethyl sulfoxide 1: 1, preparation; then connecting with a mixing bag structure; step C, precooling: the structure of the mixing bag is connected with the multi-chamber freezing bag through the interface, and the mixing bag is placed in a pre-cooling device at 4 ℃, the whole process is completed at one step, and the multi-chamber freezing storage technology is combined with the cord blood hematopoietic stem cell amplification technology, so that the cord blood can be utilized for multiple times.
Description
Technical Field
The invention relates to the field of medical treatment, in particular to a multi-chamber cryopreservation method for umbilical cord blood hematopoietic stem cells.
Background
In recent years, foreign research in the field of cord blood amplification has been rapidly advanced; before 2012, mainly cytokines, stromal cells or HOXB4 (a gene that promotes hematopoietic stem cell self-replication) were added to support expansion; from 2013, various new ideas, new theories and new products continuously emerge from HOXB4 transformation to new small molecules discovery; in the five years of 2013 and 2017, 20 clinical trials for studying umbilical cord blood cell expansion are available on average every year.
In the domestic cord blood bank, the enriched cord blood hematopoietic stem cells are mostly frozen in a single chamber, so that one part of cord blood can only be applied once; in view of the above, the present invention has been made in view of the above problems.
Disclosure of Invention
The invention provides a multi-chamber cryopreservation method for umbilical cord blood hematopoietic stem cells in order to make up for market vacancy.
The invention aims to provide a multi-chamber cryopreservation method for umbilical cord blood hematopoietic stem cells, which solves the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a multi-chamber cryopreservation method for umbilical cord blood hematopoietic stem cells comprises the following steps: primary transfer, extraction, precooling, mixing uniformly and reserving samples, exhausting, secondary transfer and reserving samples for storage:
step A, primary transfer: transferring the cord blood hematopoietic stem cells obtained by centrifugation into a mixing bag structure, mixing uniformly, reserving a sample and weighing;
and B, extraction: and (3) extracting the cryoprotectant by using a screw injector, wherein the cryoprotectant is prepared from dextran and dimethyl sulfoxide 1: 1, preparation; then connecting with a mixing bag structure;
step C, precooling: connecting the blending bag structure with a multi-chamber freezing bag through a connector, and putting the blending bag structure into a 4 ℃ precooling device;
step D, mixing uniformly and reserving samples: after precooling is finished, injecting the frozen protective agent into a mixing bag structure by using an injection pump, shaking up while infusing, and flushing a pipeline; pumping out gas and a small amount of blood in the mixing bag by utilizing a screw injector connected with the mixing bag structure, and reserving a sample in the screw injector for carrying out final product bacteria detection;
step E, exhausting: pumping out air in the freezing bag by using a self-contained syringe connected with the multi-chamber freezing bag to keep the multi-chamber freezing bag at negative pressure;
step F, secondary transfer: transferring the cord blood in the mixing bag to a freezing bag by using negative pressure in the multi-chamber freezing bag, and adjusting by using a self-contained injector connected with the multi-chamber freezing bag to enable a pipeline to be full;
step G, sample reserving and storing: the sample is retained by heat sealing the multi-chamber freezing bag pipeline and is filled into an aluminum box.
Further, in the primary transfer stage in the step A, the screw cap at the second interface of the mixing bag is unscrewed, the prepared cord blood hematopoietic stem cells in the transfer bag are sucked out by an injector, the cord blood hematopoietic stem cells are pumped into the mixing bag structure through the second interface of the mixing bag, a sample is left after shaking uniformly, and the screw cap is screwed off, and the addition amount of hydroxyethyl starch is as follows: cord blood volume: the ratio of hydroxyethyl starch is 5:1, the centrifugation is carried out for three times, the first centrifugation temperature is set to be 10 ℃, the centrifugal force is 50g, and the centrifugation time is 7 min; setting the second centrifugation temperature to 10 ℃, the centrifugal force to 420g and the centrifugation time to 7 min; the third centrifugation temperature was set at 10 deg.C, the centrifugal force was 420g, and the centrifugation time was 7 min.
And in the extraction stage in the step B, displaying numbers according to an information system by using a screw injector, extracting the cryoprotectant, unscrewing a first interface of the mixing bag, inserting the mixing bag into the screw injector, unscrewing a second interface of the mixing bag, connecting the first interface with a first interface of a freezing bag, connecting the first interface with the second interface of the freezing bag, arranging the second interface of the freezing bag with a self-contained injector, and then putting the whole system into a precooling device for precooling to 4 ℃.
And in the step D, in the sample mixing and reserving stage, a screw injector filled with DMSO is installed on an injection pump in a 4 ℃ environment, the mixing bag structure is placed on a shaking table, the mixing bag structure is shaken while being injected, the cryopreservation protective agent is injected into the mixing bag according to 1ml/min, then the mixing bag structure is repeatedly mixed for 4 times by extraction/injection at 43.11ml/min, the injection amount of each extraction is 15ml, after the DMSO injection is completed, the screw injector is used for extracting air and a small amount of blood in the mixing bag structure, the screw injector is arranged upright, the blood in the pipeline is pumped back to the mixing bag structure, a sample is reserved on the screw injector for bacteria detection, and the final concentration of the cryopreservation protective agent DMSO is 9.8%.
And in the air exhausting stage in the step E, the first pipe clamp and the second pipe clamp are closed, the self-contained syringe connected with the multi-chamber freezing bag is used for pumping air out of the multi-chamber freezing bag, and the third pipe clamp is closed, so that the multi-chamber freezing bag keeps negative pressure.
And in the second transfer stage in the step F, opening a second pipe clamp, transferring the cord blood in the mixing bag structure into the multi-chamber freezing bag, then using a self-contained injector of the multi-chamber freezing bag to extract air bubbles of the multi-chamber freezing bag and residual blood in the mixing bag structure, inverting the self-contained injector, returning the blood, and stopping after the multi-chamber freezing bag and the pipeline are full.
Furthermore, in the step G, in the sample storage stage, the pipelines of the multi-chamber freezing bag are subjected to heat combination and bar code pasting, the multi-chamber freezing bag and the pipelines are kept in a connection state, and the multi-chamber freezing bag is integrally filled into an aluminum box.
Further, the multi-chamber freezing bag tip sets up through connecting with freezing bag communicating pipe intercommunication, and install first branch pipe on the freezing bag communicating pipe, the tip of freezing bag communicating pipe passes through mixing bag second interface, freezing bag first interface and mixing bag communicating pipe intercommunication, the lower extreme and the mixing bag structure intercommunication setting of mixing bag communicating pipe, the second branch pipe is installed to the below of mixing bag communicating pipe simultaneously, install first pipe clamp on the second branch pipe, install the third pipe clamp on the first branch pipe, the second pipe clamp is installed to the afterbody of freezing bag communicating pipe, first pipe clamp, second pipe clamp and third pipe clamp are the flow regulator for the infusion hose.
Compared with the prior art, the invention has the beneficial effects that: sucking out the prepared umbilical cord blood hematopoietic stem cells in the transfer bag by using an injector, pumping into a mixing bag structure through a second interface of the mixing bag, shaking uniformly, then reserving a sample, and screwing a screw cap to be dead, wherein the addition amount of hydroxyethyl starch is as follows: cord blood volume: the ratio of hydroxyethyl starch is 5:1, and the centrifugation is carried out for three times; the mixing bag is utilized in the adding process of the freezing protective agent, so that the cells are prevented from being killed by overhigh concentration of the local protective agent when the cord blood and the freezing protective agent are mixed uniformly; the multi-chamber cryopreservation technology is combined with the cord blood hematopoietic stem cell amplification technology, so that the cord blood can be used for multiple times.
Drawings
FIG. 1 is a schematic view of a multi-chamber cryopreservation apparatus for hematopoietic stem cells according to the present invention.
In the figure: 1. a first port of the mixing bag; 2. a second port of the mixing bag; 3. a first port of the freezing bag; 4. a second port of the freezing bag; 5. a first pipe clamp; 6. a second pipe clamp; 7. a third pipe clamp; 8. a multi-chamber freezing bag; 9. a mixing bag structure; 10. a freezing bag communicating pipe; 11. a first branch pipe; 12-1, a screw injector; 12-2, self-contained syringes; 13. a second branch pipe; 14. the mixing bag is communicated with the pipe.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The first embodiment is as follows: referring to fig. 1, the present invention provides a technical solution: a multi-chamber cryopreservation method for umbilical cord blood hematopoietic stem cells comprises the following steps: primary transfer, extraction, precooling, mixing uniformly and reserving samples, exhausting, secondary transfer and reserving samples for storage:
step A, primary transfer: transferring the cord blood hematopoietic stem cells obtained by centrifugation into a mixing bag structure 9, mixing uniformly, reserving a sample and weighing;
and B, extraction: extracting cryoprotectant with a screw injector 12-1, wherein the cryoprotectant is prepared from dextran and dimethyl sulfoxide 1: 1, preparation; then is connected with a mixing bag structure 9;
step C, precooling: connecting a blending bag structure 9 with a multi-chamber freezing bag 8 through a connector, and putting the blending bag structure and the multi-chamber freezing bag into a 4 ℃ precooling device;
step D, mixing uniformly and reserving samples: after precooling is finished, injecting the frozen protective agent into the mixing bag structure 9 by using an injection pump, shaking up while infusing, and flushing a pipeline; pumping out gas and a small amount of blood in the mixing bag by using the mixing bag structure 9 connected with the screw injector 12-1, and reserving a sample in the screw injector 12-1 for carrying out final product bacteria detection;
step E, exhausting: using a self-contained syringe 12-2 connected with the multi-chamber freezing bag 8 to extract gas in the freezing bag 8, and keeping the multi-chamber freezing bag 8 at negative pressure;
step F, secondary transfer: transferring the cord blood in the mixing bag 9 to the freezing bag 8 by utilizing the negative pressure in the multi-chamber freezing bag 8, and regulating by utilizing a self-contained injector 12-2 connected with the multi-chamber freezing bag 8 to enable the pipeline to be full;
step G, sample reserving and storing: the multi-chamber freezing bag 8 is sealed in a heat seal way, and the pipe is reserved and is filled into an aluminum box.
The second embodiment is as follows: in the first transfer stage in step a, the screw cap at the second connector 2 of the mixing bag is unscrewed, the prepared umbilical cord blood hematopoietic stem cells in the transfer bag are sucked out by using an injector, the umbilical cord blood hematopoietic stem cells are driven into the mixing bag structure 9 through the second connector 2 of the mixing bag, the sample is left after uniform shaking, the screw cap is screwed off, and the addition amount of hydroxyethyl starch is as follows: cord blood volume: the ratio of hydroxyethyl starch is 5:1, the centrifugation is carried out for three times, the first centrifugation temperature is set to be 10 ℃, the centrifugal force is 50g, and the centrifugation time is 7 min; setting the second centrifugation temperature to 10 ℃, the centrifugal force to 420g and the centrifugation time to 7 min; the third centrifugation temperature was set at 10 deg.C, the centrifugal force was 420g, and the centrifugation time was 7 min.
The third concrete implementation mode: in the extraction stage in the step B, a screw injector 12-1 is used to display numbers according to an information system, the cryoprotectant is extracted, the first port 1 of the mixing bag is unscrewed and inserted into the screw injector 12-1, the second port 2 of the mixing bag and the first port 3 of the freezing bag are unscrewed and then connected, the second port 4 of the freezing bag is provided with the self-contained injector 12-2, and then the whole system is placed into a precooling device to be precooled to 4 ℃.
The fourth concrete implementation mode: in the blending sample-keeping stage in the step D, a screw injector 12-1 filled with DMSO is installed on an injection pump in an environment of 4 ℃, a blending bag structure 9 is placed on a shaking table and shaken while injecting, a cryopreservation protective agent is injected into the blending bag according to 1ml/min, then the blending bag is repeatedly mixed 4 times by extraction/injection at 43.11ml/min, the injection amount of each extraction is 15ml, after the injection of DMSO is completed, air and a small amount of blood in the blending bag structure 9 are extracted by the screw injector 12-1, the screw injector 12-1 is upright, the blood in the pipeline is pumped back to the blending bag structure 9, and a sample is kept in the screw injector 12-1 for bacteria detection, and the concentration of the cryopreservation protective agent DMSO is 9.8%.
The fifth concrete implementation mode: in the air exhaust step of the first embodiment of the present invention, the first and second tube clamps 5 and 6 are closed, the self-contained syringe 12-2 connected to the multi-chamber freezing bag 8 is used to evacuate air from the multi-chamber freezing bag 8, and the third tube clamp 7 is closed to maintain the multi-chamber freezing bag 8 at a negative pressure.
The sixth specific implementation mode: in the second transfer stage in the step F, the second tube clamp 6 is opened to transfer the cord blood in the mixing bag structure 9 into the multi-chamber freezing bag 8, then the self-contained syringe 12-2 of the multi-chamber freezing bag 8 is used to draw out the bubbles in the multi-chamber freezing bag 8 and the residual blood in the mixing bag structure 9, the self-contained syringe 12-2 is inverted to draw back the blood, and the operation is stopped after the multi-chamber freezing bag 8 and the pipeline are filled.
The seventh embodiment: in the sample storage stage in the step G, the multi-chamber freezing bag 8 is thermally combined and bar-coded, and the multi-chamber freezing bag 8 and the pipeline are kept connected and integrally placed in an aluminum box.
The specific implementation mode is eight: the seventh embodiment is further limited by the seventh embodiment, the end of the multi-chamber freezing bag 8 is communicated with the freezing bag communicating pipe 10 through a joint, the freezing bag communicating pipe 10 is provided with a first branch pipe 11, the end of the freezing bag communicating pipe 10 is communicated with the blending bag communicating pipe 14 through a blending bag second interface 2, a freezing bag first interface 3, the lower end of the blending bag communicating pipe 14 is communicated with the blending bag structure 9, meanwhile, the second branch pipe 13 is installed below the blending bag communicating pipe 14, the second branch pipe 13 is provided with a first pipe clamp 5, the first branch pipe 11 is provided with a third pipe clamp 7, the tail of the freezing bag communicating pipe 10 is provided with a second pipe clamp 6, and the first pipe clamp 5, the second pipe clamp 6 and the third pipe clamp 7 are flow regulators for infusion hoses.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A multi-chamber cryopreservation method for umbilical cord blood hematopoietic stem cells comprises the following steps: the sample is reserved to once shift, extraction, precooling, mixing, exhaust, secondary transfer and reserve the sample and deposit, its characterized in that:
step A, primary transfer: transferring the cord blood hematopoietic stem cells obtained by centrifugation into a mixing bag structure (9), mixing uniformly, and then reserving a sample and weighing;
and B, extraction: extracting cryoprotectant with a screw syringe (12-1), wherein the cryoprotectant is prepared from dextran and dimethyl sulfoxide 1: 1, preparation; then is connected with a mixing bag structure (9);
step C, precooling: connecting the blending bag structure (9) with the multi-chamber freezing bag (8) through a connector, and putting the blending bag structure and the multi-chamber freezing bag into a pre-cooling device at 4 ℃;
step D, mixing uniformly and reserving samples: after precooling is finished, injecting the frozen protective agent into a mixing bag structure (9) by using an injection pump, shaking up while infusing, and flushing a pipeline; a mixing bag structure (9) is connected with a screw injector (12-1) to pump out gas and a small amount of blood in the mixing bag, and a sample is reserved in the screw injector (12-1) to carry out final product bacteria detection;
step E, exhausting: the gas in the freezing bag (8) is extracted by using a self-contained syringe (12-2) connected with the multi-chamber freezing bag (8), so that the multi-chamber freezing bag (8) keeps negative pressure.
Step F, secondary transfer: transferring the cord blood in the mixing bag (9) to the freezing bag (8) by utilizing the negative pressure in the multi-chamber freezing bag (8), and adjusting by utilizing a self-contained injector (12-2) connected with the multi-chamber freezing bag (8) to enable the pipeline to be full;
step G, sample reserving and storing: the pipe of the heat-seal multi-chamber freezing bag (8) is reserved and is filled into an aluminum box.
2. The method for multi-compartment cryopreservation of umbilical cord blood hematopoietic stem cells according to claim 1, wherein: in the primary transfer stage in the step A, unscrewing a screw cap at the second interface (2) of the mixing bag, sucking out the prepared umbilical cord blood hematopoietic stem cells in the transfer bag by using an injector, pumping the umbilical cord blood hematopoietic stem cells into a mixing bag structure (9) through the second interface (2) of the mixing bag, shaking uniformly, reserving a sample, and screwing the screw cap to death, wherein the addition amount of hydroxyethyl starch is as follows: cord blood volume: the ratio of hydroxyethyl starch is 5:1, the centrifugation is carried out for three times, the first centrifugation temperature is set to be 10 ℃, the centrifugal force is 50g, and the centrifugation time is 7 min; setting the second centrifugation temperature to 10 ℃, the centrifugal force to 420g and the centrifugation time to 7 min; the third centrifugation temperature was set at 10 deg.C, the centrifugal force was 420g, and the centrifugation time was 7 min.
3. The method for multi-compartment cryopreservation of umbilical cord blood hematopoietic stem cells according to claim 1, wherein: and in the extraction stage in the step B, displaying numbers according to an information system by using a screw injector (12-1), extracting the cryoprotectant, unscrewing a first port (1) of the mixing bag, inserting the mixing bag into the screw injector (12-1), unscrewing a second port (2) of the mixing bag, connecting the first port (3) of the freezing bag with a second port (4) of the freezing bag, placing the whole system into a precooling device, and precooling to 4 ℃.
4. The method for multi-compartment cryopreservation of umbilical cord blood hematopoietic stem cells according to claim 1, wherein: and D, in the sample mixing and reserving stage, a screw injector (12-1) filled with DMS O is installed on an injection pump under the environment of 4 ℃, a mixing bag structure (9) is placed on a shaking table, the shaking table is carried out while the injection is carried out, the cryopreservation protective agent is injected into the mixing bag according to 1ml/min, then the mixing bag structure is extracted/injected for repeated mixing 4 times at 43.11ml/min, the injection amount of each extraction is 15ml, after the DMSO injection is finished, air and a small amount of blood in the mixing bag structure (9) are extracted by the screw injector (12-1), the screw injector (12-1) is rightly placed, the blood in the pipeline is pumped back to the mixing bag structure (9), the sample is reserved on the screw injector (12-1) for bacteria detection, and the final concentration of the cryopreservation protective agent DMSO is 9.8%.
5. The method for multi-compartment cryopreservation of umbilical cord blood hematopoietic stem cells according to claim 1, wherein: and in the air exhausting stage in the step E, the first pipe clamp (5) and the second pipe clamp (6) are closed, air in the multi-chamber freezing bag (8) is exhausted by using a self-contained syringe (12-2) connected with the multi-chamber freezing bag (8), and the third pipe clamp (7) is closed to enable the multi-chamber freezing bag (8) to keep negative pressure.
6. The method for multi-compartment cryopreservation of umbilical cord blood hematopoietic stem cells according to claim 1, wherein: and in the secondary transfer stage in the step F, the second pipe clamp (6) is opened, the cord blood in the mixing bag structure (9) is transferred into the multi-chamber freezing bag (8), then the self-contained injector (12-2) of the multi-chamber freezing bag (8) is used for pumping out air bubbles of the multi-chamber freezing bag (8) and residual blood in the mixing bag structure (9), the self-contained injector (12-2) is inverted, the blood is pumped back, and the mixing bag structure is stopped after the multi-chamber freezing bag (8) and the pipeline are full.
7. The method for multi-compartment cryopreservation of umbilical cord blood hematopoietic stem cells according to claim 1, wherein: and G, in the sample storage stage, the pipelines of the multi-chamber freezing bag (8) are subjected to heat combination and bar code pasting, the multi-chamber freezing bag (8) and the pipelines are kept in a connection state, and the multi-chamber freezing bag is integrally filled into an aluminum box.
8. The method for multi-compartment cryopreservation of umbilical cord blood hematopoietic stem cells according to claim 7, wherein: the multi-chamber freezing bag (8) tip sets up through connecting with freezing bag communicating pipe (10) intercommunication, and install first branch pipe (11) on freezing bag communicating pipe (10), the tip of freezing bag communicating pipe (10) is through mixing bag second interface (2), freezing bag first interface (3) and mixing bag communicating pipe (14) intercommunication, the lower extreme and mixing bag structure (9) intercommunication setting of mixing bag communicating pipe (14), second branch pipe (13) are installed to the below of mixing bag communicating pipe (14) simultaneously, install first pipe clamp (5) on second branch pipe (13), install third pipe clamp (7) on first branch pipe (11), second pipe clamp (6) are installed to the afterbody of freezing bag communicating pipe (10), first pipe clamp (5), second pipe clamp (6) and third pipe clamp (7) are the flow regulator that the infusion hose was used.
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| CN115486440A (en) * | 2022-10-20 | 2022-12-20 | 许艳杰 | Integrated into one piece connects cryopreserved bag verifying attachment |
| CN115486441A (en) * | 2022-10-28 | 2022-12-20 | 许艳杰 | Freezing storage mixing three-dimensional bag and use method |
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Application publication date: 20211112 |