CN113046305A - Separation and purification method of islet cells - Google Patents
Separation and purification method of islet cells Download PDFInfo
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- CN113046305A CN113046305A CN202110476092.0A CN202110476092A CN113046305A CN 113046305 A CN113046305 A CN 113046305A CN 202110476092 A CN202110476092 A CN 202110476092A CN 113046305 A CN113046305 A CN 113046305A
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Abstract
The invention relates to the technical field of cell separation, and discloses a method for separating and purifying islet cells, which comprises the following steps: 1) suturing and ligating the interface of the tail part of the perfusion needle and the pancreatic duct, and perfusing the mixed enzyme into the pancreas through the perfusion needle; 2) digesting the pancreas and collecting pancreatic tissue fluid; during the digestion treatment, sampling, microscopic examination and evaluation were performed to assess the attainment of collection conditions: the proportion of free islet cells is more than 50%, and pancreatic tissue fluid begins to be collected; 3) centrifuging pancreatic tissue fluid, collecting precipitate, and adding purified solution and albumin to obtain tissue suspension; 4) the tissue suspension was subjected to gradient centrifugation at about 4 ℃ to collect islet cells. The separation and purification method provided by the invention has the advantages that the perfusion time is shortened, the optimal time for collecting pancreatic tissue fluid is mastered, and the gradient centrifugal separation is carried out at low temperature, so that the survival rate and the form of islet cells are protected, and the final islet cell separation quantity, survival rate and purity are improved.
Description
Technical Field
The invention relates to the technical field of cell separation, in particular to a method for separating and purifying islet cells.
Background
The islets of langerhans are the endocrine glands of the pancreas, whose main role is to sense and regulate the blood glucose levels in the body. Islets are morphologically a large number of cell clusters of varying size and shape, with the major cell types including 5 cells, α, β, δ, PP, and e. The pancreatic islet alpha cells (about 24% -40%) secrete glucagon to promote the increase of blood sugar level in vivo, while the pancreatic islet beta cells (about 60% -80%) secrete insulin to reduce the blood sugar level in vivo; the islet delta cells (about 6-15%) secrete somatostatin, and hormone secretion of alpha cells and beta cells is influenced in a negative feedback mode to regulate blood sugar balance; pancreatic islet PP cells secrete pancreatic polypeptide, and islet epsilon cells secrete ghrelin.
Type 1 diabetes is a type of diabetes caused by islet cell destruction and absolute deficiency of insulin secretion, and requires lifelong insulin therapy. The most ideal method for treating diabetes (especially type 1 diabetes) is cell therapy, i.e. islet cells are isolated from pancreas and tested and treated accordingly. Islet cells used in cell therapy are derived from donated human or porcine pancreas and are isolated from the donor pancreas to obtain high quality living cells. However, the yield, the survival rate and the purity of the obtained pancreatic islets are low by the current pancreatic islet separating and preparing technology.
Disclosure of Invention
The invention aims to provide a method for separating and purifying islet cells, which solves one or more technical problems in the prior art and provides at least one beneficial selection or creation condition.
The technical scheme adopted for solving the technical problems is as follows:
a method for separating and purifying islet cells comprises the following steps:
1) obtaining a pancreas of a donor, suturing and ligating an interface of the tail part of a perfusion needle and a pancreatic duct of the pancreas, and perfusing mixed enzyme into the pancreas through the perfusion needle; the donor is selected to be a human donor.
2) Dividing the perfused pancreas into a plurality of pancreas blocks, mixing the pancreas blocks with digestive juice, performing digestion treatment, and collecting pancreas tissue fluid; sampling, microscopic examination and evaluation are carried out at intervals in the digestion treatment process, and pancreatic tissue fluid is collected after the evaluation reaches the collection condition; the collection conditions include: the proportion of free islet cells is greater than 50%;
3) centrifuging the pancreatic tissue fluid, collecting precipitates, adding the precipitates into a purified solution, and adding human serum albumin for mixing to obtain a tissue suspension;
4) and adding the tissue suspension into a density gradient separation solution, performing gradient centrifugation at the temperature of about 4 ℃, and collecting islet cells.
Preferably, in step 2), the specific process of the digestion treatment is as follows: putting a plurality of pancreas blocks into a digestion device, introducing the digestion solution, digesting at 32-38 ℃, sampling every 1-5 minutes, dyeing the sample by adopting dithizone, observing under an inverted microscope, evaluating and observing the digestion condition, reducing the temperature to 20-30 ℃ to continue digesting by combining the digestion degree of pancreas tissues in a digestion tank, starting to collect pancreas tissue solution, collecting pancreas tissue solution and digesting simultaneously, and stopping digestion when the digestion is complete.
Preferably, in step 2), the collection conditions are: the tissue coverage in the microscopic field is greater than 50%, the number of islet cells is greater than 45 and the proportion of islet cell debris is less than 10%.
The number of islet tissues and the number of islet cells are both visually observed by observing the sample with a microscope, the sample is gathered to the center of the culture dish, and the sample is observed with an inverted microscope to estimate the number of tissues and the number of islet cells.
The proportion of free islet cells is: the proportion of free islet cells to the total number of islet cells; free islet cells means that less than 25% of the islet cells are marginally attached to the acinar tissue.
The proportion of islet cell fragments refers to the proportion of islet cell fragments to the total number of islet cells; islet cell debris refers to those islet cells that are damaged by excessive digestion, resulting in ragged borders.
Preferably, in step 1), raw materials for preparing the mixed enzyme solution comprise neutral protease and collagenase; the specific process for obtaining the pancreas of the donor is as follows: the connective tissue around the pancreas was removed, and the connecting port between the pancreatic duct and the duodenum of the pancreas was ligated and transected from the pancreatic neck of the pancreas to obtain a pancreas with an intact envelope. The pancreas capsule was left intact to prevent leakage of mixed enzyme solution during perfusion.
Preferably, in step 1), the specific process of perfusion is as follows: inserting a perfusion needle into a main pancreatic duct of the pancreas, suturing and ligating an interface of the tail of the perfusion needle and the pancreatic duct by using an operation needle line, sucking the mixed enzyme solution by using an injector, connecting the mixed enzyme solution with the perfusion needle, and pressurizing and perfusing the pancreas until the pancreas is fully expanded, thus stopping perfusion.
The pancreas adopts the fixed operation method of stitching ligature with the afterbody of filling the needle and the kneck of pancreatic duct during filling, is difficult for the backward flow seepage, and whole filling flow needs 20 ~ 30 minutes, reduces the ischemia of islet cells, oxygen deficiency time, keeps islet cells's survival rate better.
Preferably, in step 2), before the operation of dividing the perfused pancreas into a plurality of pancreas pieces, the capsule of the perfused pancreas needs to be removed. After the perfusion is finished, the capsule of the pancreas needs to be trimmed and cleaned up, so that the situation that the capsule blocks a filter screen of a digestion tank during subsequent digestion is avoided, and the digestion quality is influenced.
Preferably, in step 3), the specific process of centrifugation is as follows: centrifuging the pancreatic tissue fluid for 1-3 min at 800-1200 rpm and 2-8 ℃, adding the obtained precipitate into washing liquor, centrifuging again, and repeatedly centrifuging for several times.
Preferably, in the step 4), the density gradient separation liquid has a density difference of 1.06-1.10g/cm3With continuous density gradient separation betweenAnd (4) liquid.
Preferably, formulating the density gradient separation fluid comprises the following steps: the density was 1.06g/cm3Has a low density gradient separation liquid and a density of 1.10g/cm3High density gradient separation of liquids.
Preferably, in step 2), the size of the pancreatic block is a small block with a side of about 2 cm.
Compared with the prior art, the invention has the following beneficial effects:
according to the method for separating and purifying the islet cells, the operation method of suturing, ligating and fixing the joint of the tail part of the perfusion needle and the pancreatic duct is adopted during pancreas perfusion, so that backflow leakage is not easy to occur, the perfusion time is shortened, the ischemia and anoxia time of the islet cells is shortened, and the survival rate of the islet cells is better reserved; sampling, microscopic examination and evaluation are carried out in the process of digestion treatment, and after the collection condition that the proportion of free islet cells is more than 50% is evaluated, the collection of pancreatic tissue fluid is started, so that the optimal time for collecting pancreatic tissue fluid can be accurately grasped, and the problems of low islet cell separation quantity and low survival rate caused by insufficient digestion or excessive digestion are solved; by performing gradient centrifugation at a low temperature of about 4 ℃, the survival rate and morphology of islet cells can be protected, and the stability of purification and the quality of islet cells can be improved.
Drawings
The invention is further described below with reference to the accompanying drawings and examples.
FIG. 1 is a schematic diagram of PI/FDA co-staining of islet cells obtained in example 1 of the present invention;
FIG. 2 is a schematic diagram of the islet cells obtained in example 1 of the present invention stained with dithizone;
FIG. 3 is a schematic diagram of PI/FDA co-staining of islet cells obtained in comparative example 1 of the present invention;
FIG. 4 is a schematic diagram of the islet cells obtained in comparative example 1 of the present invention stained with dithizone.
Detailed Description
The conception and the technical effects of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments, so that the objects, features and effects of the present invention can be fully understood. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
The reagents and equipment used in the following examples are commercially available.
The main reagents are as follows:
mixing enzyme solution: is prepared from 200IU of neutral protease and collagenase.
Digestion solution: is prepared from Phase I Solution and heparin sodium injection (the final concentration is 10U/mL).
High density gradient separation: is prepared from Cold Storage/Purification Stock Solution, Penta Starch 10% Solution, heparin sodium injection, and Gradient Stock Solution.
Low density gradient separation: is prepared from Cold Storage/Purification Storage Solution, Penta Starch 10% Solution, heparin sodium injection and separating medium Optiprep.
Washing liquid: is prepared from Cold Storage/Purification Storage Solution, Penta Starch 10% Solution, heparin sodium injection (with the final concentration of 10U/mL) and human serum albumin injection (with the final concentration of 6.25 g/L).
Purifying the solution: is prepared from Cold Storage/Purification Storage Solution, Penta Starch 10% Solution and heparin sodium injection (the final concentration is 10U/mL).
A neutral protease source Nordmark/S3030112; a collagenase source Nordmark/S1745503; phase I Solution source Cellgro/99-678-CM, Cold Storage/Purification Storage Solution source Cellgro/99-677-CM, Penta Starch 10% Solution source Cellgro/99-723-CM, Gradient Storage Solution source Cellgro/99-674-CM; the separated liquid Optiprep is from Axis-shield company.
The main apparatus comprises: a perfusion needle, a syringe, a surgical instrument, a peristaltic pump, a temperature display, a water bath, a digestion vessel (brand: biornep RICORDI model: 500ml), an inverted microscope, a conical centrifuge tube, a low-speed refrigerated centrifuge, a COBE 2991 cell separator, a COBE bag, and a gradient mixer (brand biornep).
The purity of the islet cells is detected by the following indexes:
1. the purity of the islets means: islet counts were performed using Dithizone (DTZ) staining. Islet purity is the ratio of the number of islets that stained positive for DTZ to the total number of purified cell masses. Is used for reflecting the purification effect of the pancreatic island and reflecting the index of the technical level of the pancreatic island separation and purification.
2. Islet equivalent means: islet Equivalent (IEQ) is an Islet counting method, where 1 Islet equivalent is a 150 μm diameter Islet. Total islet equivalents refer to the total number of islet equivalents in the sample. Is used for calculating and obtaining the number of the pancreatic islets and reflecting the index of the technical level of pancreatic islet separation and purification.
3. The islet survival rate refers to: islet counts were performed using live cell staining techniques. The islet survival rate is the ratio of the number of viable islets to the total number of islets. Is used for evaluating and obtaining the proportion of living cells in the pancreatic islets and embodies the index of the technical level of pancreatic islet separation and purification.
Example 1
A method for separating and purifying islet cells comprises the following steps:
1) placing the pancreas of a human donor into a deep tray containing a Trimming solution (1g/L cefazolin sodium), removing other connecting tissues, keeping the envelope of the pancreas intact, ligating a connecting port between a pancreatic duct and duodenum, crossing the trimmed pancreatic neck, finding the position of a main pancreatic duct, inserting a perfusion needle, and suturing and ligating the interface between the tail of the perfusion needle and the pancreatic duct by using an operation needle thread; and sucking the mixed enzyme solution by using a 50mL screw injector, manually pressurizing and injecting the mixed enzyme solution on an injection needle head in a screwing mode, stopping injecting when the pancreas is fully expanded by naked eyes, and trimming for the second time to remove the capsule and residual non-pancreatic tissues.
2) The pancreas fully-inflated by perfusion is cut into small blocks with the side length of about 2 centimeters, the small blocks and the residual mixed enzyme solution are placed into a digestion tank, a peristaltic pump is started to introduce digestion liquid, circulation is formed in the digestion tank, the digestion tank is shaken at the same time, the digestion temperature is controlled to be 37 ℃, the digestion condition of the pancreas blocks in a digestion device is observed, when the pancreas tissue structure begins to become loose, the digestion liquid becomes turbid, samples are taken at intervals of 5 minutes, and the sampling interval time is gradually shortened to 1 minute along with the digestion process. Adding dithizone into the sample for dyeing, observing under an inverted microscope, and evaluating; when the coverage proportion of the tissue in a microscopic field is observed to be more than 50%, the number of islet cells is more than 45, the proportion of free islet cells is more than 50% and the proportion of islet cell fragments is less than 10%, the digestion temperature is gradually reduced to 30 ℃ in combination with the observation of the digestion degree of the pancreatic tissue in the digestion tank, the collection of pancreatic tissue liquid by the collection bottle is started while the digestion is continued, in the process of collecting the pancreatic tissue liquid, RPMI1640 supplementary circulation is simultaneously introduced into the digestion tank to ensure that the pancreatic tissue is fully digested, when the collection bottle reaches 1L, the next bottle is collected, the collected bottle is placed on crushed ice, and the next bottle is subjected to subpackaging and centrifugation.
3) When pancreas tissue digestion is completely observed and evaluated, collecting all pancreas tissue fluid, subpackaging the pancreas tissue fluid into a plurality of conical centrifuge bottles, centrifuging for 2min at 1000rpm and 4 ℃, discarding all supernatant fluid, mixing precipitates in the conical centrifuge bottles, adding washing liquor into the conical centrifuge bottles, centrifuging and washing for several times, gradually reducing the number of the conical centrifuge bottles in the washing process, finally aggregating the precipitates into one conical centrifuge bottle, adding corresponding volume of purified liquid according to the volume of the precipitates in the conical centrifuge bottles for resuspension (if the volume of the precipitates is less than or equal to 25mL, adding 100mL of purified liquid, if the volume of the precipitates is more than 25mL, adding 200mL of purified liquid, equally dividing for twice purification), adding 20mL of human serum albumin, uniformly mixing, and ensuring that the precipitates are not coagulated into blocks to obtain tissue suspension.
4) The COBE bag is arranged in a COBE 2991 cell separator, a pipeline is connected, the COBE 2991 cell separator is connected with a water chiller, the temperature of a centrifugal tank of the COBE 2991 cell separator is kept at about 4 ℃, a peristaltic pump is started to start to introduce 120mL of the cells with the density of 1.10g/cm3After the introduction of the high-density gradient separation solution is finished, the peristaltic pump is stopped, and COBE 2991 cells are pressed for separationThe START-SPIN button of the machine STARTs to operate, the SUPER-OUT button is pressed after 20 seconds to exhaust air in the bag, and the STOP-RESET button is pressed to STOP the machine from rotating; 120mL of a 1.10g/cm density solution were added to the left cup of the gradient mixer3The right cup is added with 120mL of high density gradient separation solution with the density of 1.06g/cm3Opening the intermediate valve, properly mixing to obtain the density gradient separating liquid, starting a START-SPIN button, starting a peristaltic pump for introduction, and establishing a continuous density gradient surface in the COBE bag.
5) And (3) when the introduction is finished and the last liquid is remained in the pipeline and is connected with a gradient mixer, adding the tissue suspension obtained in the step 3) into the left cup of the gradient mixer, recording the time for 1min for 20s, stopping the peristaltic pump for 30s, starting the peristaltic pump for 30s, circularly operating the peristaltic pump for 30s, starting the peristaltic pump for 30s, slowly and completely introducing the tissue suspension into the COBE bag, adding 35mL of purified liquid for rinsing when a very small amount of cell suspension is remained in the gradient mixer, and then continuously introducing into the COBE bag to ensure that no tissue suspension remains in the gradient mixer. After all the cells are introduced, stopping the peristaltic pump, and idling the COBE 2991 cell separator for 4min to ensure that the density gradient surface is distributed more uniformly; and during the waiting period, preparing a purification collection bottle, adding 225mL of CMRL 1066 culture medium into the 250mL purification collection bottle, pressing a SOPER-OUT button to collect the effluent after the idle time is over, and pressing a STOP-RESET button to STOP the machine from rotating after the complete collection is finished, thus obtaining the separated and purified islet cells.
An appropriate amount of the islet cells separated and purified in example 1 was used as a sample for purity detection. As shown in fig. 1, the survival rate of islet cells (PI/FDA co-staining) > 98%; as shown in fig. 2, the number of islet cells was about 8000 IEQ/gram of pancreas, with a total purity (DTZ staining) of about 85%.
Example 2
A method for separating and purifying islet cells comprises the following steps:
1) placing the pancreas of a human donor into a deep tray containing a Trimming solution (1g/L cefazolin sodium), removing other connecting tissues, keeping the envelope of the pancreas intact, ligating a connecting port between a pancreatic duct and duodenum, crossing the trimmed pancreatic neck, finding the position of a main pancreatic duct, inserting a perfusion needle, and suturing and ligating the interface between the tail of the perfusion needle and the pancreatic duct by using an operation needle thread; and sucking the mixed enzyme solution by using a 50mL screw injector, manually pressurizing and injecting the mixed enzyme solution on an injection needle head in a screwing mode, stopping injecting when the pancreas is fully expanded by naked eyes, and trimming for the second time to remove the capsule and residual non-pancreatic tissues.
2) The pancreas fully-inflated by perfusion is cut into small blocks with the side length of about 2 centimeters, the small blocks and the residual mixed enzyme solution are placed into a digestion tank, a peristaltic pump is started to introduce digestion liquid, circulation is formed in the digestion tank, the digestion tank is shaken simultaneously, the digestion temperature is controlled to be 35 ℃, the digestion condition of the pancreas blocks in a digestion device is observed, when the pancreas tissue structure begins to become loose, the digestion liquid is turbid, samples are taken every 5 minutes, and the sampling interval time is gradually shortened to 1 minute along with the digestion process. Adding dithizone into the sample for dyeing, observing under an inverted microscope, and evaluating; when the coverage proportion of the tissue in a microscope visual field is observed to be more than 50%, the number of islet cells is more than 45, the proportion of free islet cells is more than 40% and the proportion of islet cell fragments is less than 10%, the digestion temperature is reduced to 25 ℃ in combination with the observation of the digestion degree of the pancreatic tissue in the digestion tank, the collection of pancreatic tissue liquid by the collection bottle is started while the digestion is continued, in the process of collecting the pancreatic tissue liquid, RPMI1640 supplementary circulation is simultaneously introduced into the digestion tank to ensure that the pancreatic tissue is fully digested, when the collection bottle reaches 1L, the next bottle is collected, the collected bottle is placed on crushed ice, and the bottle is subjected to subpackaging and centrifugation.
3) When pancreas tissue digestion is completely observed and evaluated, collecting all pancreas tissue fluid, subpackaging the pancreas tissue fluid into a plurality of conical centrifuge bottles, centrifuging for 1min at 800rpm and 2 ℃, discarding all supernatant fluid, mixing precipitates in the conical centrifuge bottles, adding washing liquor into the conical centrifuge bottles, centrifuging and washing for several times, gradually reducing the number of the conical centrifuge bottles in the washing process, finally aggregating the precipitates into one conical centrifuge bottle, adding corresponding volume of purified liquid according to the volume of the precipitates in the conical centrifuge bottles for resuspension (if the volume of the precipitates is less than or equal to 25mL, adding 100mL of purified liquid, if the volume of the precipitates is more than 25mL, adding 200mL of purified liquid, equally dividing for twice purification), adding 20mL of human serum albumin, uniformly mixing, and ensuring that the precipitates are not coagulated into blocks to obtain tissue suspension.
4) The COBE bag is arranged in a COBE 2991 cell separator, a pipeline is connected, the COBE 2991 cell separator is connected with a water chiller, the temperature of a centrifugal tank of the COBE 2991 cell separator is kept at about 4 ℃, a peristaltic pump is started to start to introduce 120mL of the cells with the density of 1.10g/cm3After the introduction of the high-density gradient separation liquid is finished, stopping a peristaltic pump, pressing a START-SPIN button of a COBE 2991 cell separator to START running, pressing an SUPER-OUT button after 20 seconds to discharge air in the bag, and pressing a STOP-RESET button to STOP the machine from rotating; 120mL of a 1.10g/cm density solution were added to the left cup of the gradient mixer3The right cup is added with 120mL of high density gradient separation solution with the density of 1.06g/cm3Opening the intermediate valve, properly mixing to obtain the density gradient separating liquid, starting a START-SPIN button, starting a peristaltic pump for introduction, and establishing a continuous density gradient surface in the COBE bag.
5) And (3) when the introduction is finished and the last liquid is remained in the pipeline and is connected with a gradient mixer, adding the tissue suspension obtained in the step 3) into the left cup of the gradient mixer, recording the time for 1min for 20s, stopping the peristaltic pump for 40s, starting the peristaltic pump for 40s, circularly operating the peristaltic pump for 40s, stopping the peristaltic pump for 40s, slowly and completely introducing the tissue suspension into the COBE bag, adding 35mL of purified liquid for rinsing when a very small amount of cell suspension is remained in the gradient mixer, and then continuously introducing into the COBE bag to ensure that no tissue suspension remains in the gradient mixer. After all the cells are introduced, stopping the peristaltic pump, and idling the COBE 2991 cell separator for 4min to ensure that the density gradient surface is distributed more uniformly; and during the waiting period, preparing a purification collection bottle, adding 225mL of CMRL 1066 culture medium into the 250mL purification collection bottle, pressing a SOPER-OUT button to collect the effluent after the idle time is over, and pressing a STOP-RESET button to STOP the machine from rotating after the complete collection is finished, thus obtaining the separated and purified islet cells.
Example 3
A method for separating and purifying islet cells comprises the following steps:
1) placing the pancreas of a human donor into a deep tray containing a Trimming solution (1g/L cefazolin sodium), removing other connecting tissues, keeping the envelope of the pancreas intact, ligating a connecting port between a pancreatic duct and duodenum, crossing the trimmed pancreatic neck, finding the position of a main pancreatic duct, inserting a perfusion needle, and suturing and ligating the interface between the tail of the perfusion needle and the pancreatic duct by using an operation needle thread; and sucking the mixed enzyme solution by using a 50mL screw injector, manually pressurizing and injecting the mixed enzyme solution on an injection needle head in a screwing mode, stopping injecting when the pancreas is fully expanded by naked eyes, and trimming for the second time to remove the capsule and residual non-pancreatic tissues.
2) The pancreas fully-inflated by perfusion is cut into small blocks with the side length of about 2 centimeters, the small blocks and the residual mixed enzyme solution are placed into a digestion tank, a peristaltic pump is started to introduce digestion liquid, circulation is formed in the digestion tank, the digestion tank is shaken at the same time, the digestion temperature is controlled to be 32 ℃, the digestion condition of the pancreas blocks in a digestion device is observed, when the pancreas tissue structure begins to become loose, the digestion liquid is turbid, samples are taken every 5 minutes, and the sampling interval time is gradually shortened to 1 minute along with the digestion process. Adding dithizone into the sample for dyeing, observing under an inverted microscope, and evaluating; when the coverage proportion of the tissue in a microscopic field is observed to be more than 50%, the number of islet cells is more than 45, the proportion of free islet cells is more than 30% and the proportion of islet cell fragments is less than 10%, reducing the digestion temperature to 25 ℃, starting to collect pancreatic tissue liquid by using a collecting bottle while continuing to digest, simultaneously introducing RPMI1640 supplementary circulation into a digestion tank in the process of collecting the pancreatic tissue liquid so as to ensure that the pancreatic tissue is fully digested, collecting the next bottle when the collecting bottle reaches 1L, placing the collected bottle on crushed ice, and performing subpackaging and centrifugation.
3) When pancreas tissue digestion is completely observed and evaluated, collecting all pancreas tissue fluid, subpackaging the pancreas tissue fluid into a plurality of conical centrifuge bottles, centrifuging for 3min at 1200rpm and 8 ℃, discarding all supernatant fluid, mixing precipitates in the conical centrifuge bottles, adding washing liquor into the conical centrifuge bottles, centrifuging and washing for several times in such a way, gradually reducing the number of the conical centrifuge bottles in the washing process, finally aggregating the precipitates into one conical centrifuge bottle, adding corresponding volume of purified liquid according to the volume of the precipitates in the conical centrifuge bottles for resuspension (if the volume of the precipitates is less than or equal to 25mL, adding 100mL of purified liquid, if the volume of the precipitates is more than 25mL, adding 200mL of purified liquid, equally dividing for two times of purification), adding 20mL of human serum albumin, uniformly mixing, and ensuring that the human serum albumin is not coagulated into blocks to obtain tissue suspension.
4) The COBE bag is arranged in a COBE 2991 cell separator, a pipeline is connected, the COBE 2991 cell separator is connected with a water chiller, the temperature of a centrifugal tank of the COBE 2991 cell separator is kept at about 4 ℃, a peristaltic pump is started to start to introduce 120mL of the cells with the density of 1.10g/cm3After the introduction of the high-density gradient separation liquid is finished, stopping a peristaltic pump, pressing a START-SPIN button of a COBE 2991 cell separator to START running, pressing an SUPER-OUT button after 20 seconds to discharge air in the bag, and pressing a STOP-RESET button to STOP the machine from rotating; 120mL of a 1.10g/cm density solution were added to the left cup of the gradient mixer3The right cup is added with 120mL of high density gradient separation solution with the density of 1.06g/cm3Opening the intermediate valve, properly mixing to obtain the density gradient separating liquid, starting a START-SPIN button, starting a peristaltic pump for introduction, and establishing a continuous density gradient surface in the COBE bag.
5) And (3) when the introduction is finished and the last liquid is remained in the pipeline and is connected with a gradient mixer, adding the tissue suspension obtained in the step 3) into the left cup of the gradient mixer, recording the time for 1min for 20s, stopping the peristaltic pump for 30s, starting the peristaltic pump for 30s, circularly operating the peristaltic pump for 30s, starting the peristaltic pump for 30s, slowly and completely introducing the tissue suspension into the COBE bag, adding 35mL of purified liquid for rinsing when a very small amount of cell suspension is remained in the gradient mixer, and then continuously introducing into the COBE bag to ensure that no tissue suspension remains in the gradient mixer. After all the cells are introduced, stopping the peristaltic pump, and idling the COBE 2991 cell separator for 4min to ensure that the density gradient surface is distributed more uniformly; and during the waiting period, preparing a purification collection bottle, adding 225mL of CMRL 1066 culture medium into the 250mL purification collection bottle, pressing a SOPER-OUT button to collect the effluent after the idle time is over, and pressing a STOP-RESET button to STOP the machine from rotating after the complete collection is finished, thus obtaining the separated and purified islet cells.
Comparative example 1 (different from example 1 in that the digestion temperature was started to be lowered when the collection condition was not achieved during the digestion treatment, i.e., the proportion of free islet cells was 40%)
Taking a proper amount of the separated and purified islet cells in the comparative example 1 as a sample for purity detection. As shown in FIG. 3, the survival rate of islet cells (PI/FDA co-staining) is 80-90%; as shown in FIG. 4, the number of islet cells was about 5000 IEQ/gram of pancreas, with a total purity (DTZ staining) of about 60%.
Comparative example 2 (different from example 1 in that the temperature of the centrifuge tank of the COBE 2991 cell separator was maintained at more than 20 ℃ during the purification process.)
Taking a proper amount of the separated and purified islet cells in the comparative example 2 as a sample for purity detection. The survival rate of islet cells (PI/FDA co-staining) is 80-90%; the number of islet cells was about 5000 IEQ/gram of pancreas, with a total purity (DTZ staining) of about 55%.
Comparative example 3 (different from example 1 in that the junction between the needle body of the perfusion needle and the pancreatic duct was ligated with an operation needle thread.)
Taking a proper amount of separated and purified islet cells in the comparative example 3 as a sample for purity detection. The survival rate of islet cells (PI/FDA co-staining) is 75-85%; the number of islet cells was about 5000 IEQ/gram of pancreas, with a total purity (DTZ staining) of about 55%.
From the results of the purity tests of islet cells of example 1 and comparative examples 1-3, it can be seen that the survival rate, quantity, and purity of islet cells obtained according to the present invention are significantly higher than those of islet cells obtained according to comparative examples 1-3. Comparative example 1 because the digestion temperature was lowered without reaching the collection conditions, the digestion was insufficient, and finally the survival rate, number and purity of islet cells were low; comparative example 2 because the temperature was higher during gradient centrifugation, the survival rate, number and purity of the final islet cells were also affected; comparative example 3 ligation at the interface between the needle body of the perfusion needle and the pancreatic duct causes the needle tube to move in the pancreatic duct, easily causes leakage, causes long perfusion time, prolongs the ischemia and anoxia time of islet cells, and has insufficient perfusion, and finally the survival rate, the number and the purity of the islet cells are low.
While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that the present invention is not limited to the details of the embodiments shown and described, but is capable of numerous equivalents and substitutions without departing from the spirit of the invention as set forth in the claims appended hereto.
Claims (10)
1. A method for separating and purifying islet cells is characterized by comprising the following steps:
1) suturing and ligating the interface of the tail part of the perfusion needle and the pancreatic duct, and perfusing the mixed enzyme into the pancreas through the perfusion needle;
2) dividing the perfused pancreas into a plurality of pancreas blocks, mixing the pancreas blocks with digestive juice, performing digestion treatment, and collecting pancreas tissue fluid; sampling, microscopic examination and evaluation are carried out at intervals in the digestion treatment process, and pancreatic tissue fluid is collected after the evaluation reaches the collection condition; the collection conditions include: the proportion of free islet cells is greater than 50%;
3) centrifuging the pancreatic tissue fluid, collecting precipitates, adding the precipitates into a purified solution, and adding albumin for mixing to obtain a tissue suspension;
4) and adding the tissue suspension into a density gradient separation solution, performing gradient centrifugation at the temperature of about 4 ℃, and collecting islet cells.
2. The separation and purification method according to claim 1, wherein in the step 2), the digestion treatment comprises the following specific processes: putting a plurality of pancreas blocks into a digestion device, introducing the digestion solution, digesting at 32-38 ℃, sampling every 1-5 min, dyeing the sample by adopting dithizone, observing under an inverted microscope, reducing the temperature to 20-30 ℃ after the collection condition is reached, continuing to digest, starting to collect pancreas tissue fluid, and stopping digestion when the pancreas tissue fluid is completely digested.
3. The separation and purification method according to claim 1, wherein in step 2), the collection conditions further comprise: the tissue coverage in the microscopic field is greater than 50%, the number of islet cells is greater than 45 and the proportion of islet cell debris is less than 10%.
4. The separation and purification method according to claim 1, wherein the raw materials for preparing the mixed enzyme solution in step 1) include neutral protease and collagenase.
5. The separation and purification method according to claim 1, wherein in step 1), the perfusion process comprises: inserting the perfusion needle into the pancreatic duct of the pancreas, suturing and ligating the interface of the tail part of the perfusion needle and the pancreatic duct, sucking the mixed enzyme solution by using an injector, connecting the mixed enzyme solution with the perfusion needle, and performing pressurized perfusion on the pancreas until the pancreas is fully expanded.
6. The method according to claim 1, wherein in step 2), before the step of dividing the perfused pancreas into a plurality of pancreas pieces, the envelope of the perfused pancreas needs to be removed.
7. The separation and purification method according to claim 1, wherein in the step 3), the centrifugation is performed by: centrifuging the pancreatic tissue fluid for 1-3 min at 800-1200 rpm and 2-8 ℃, adding the obtained precipitate into washing liquor, centrifuging again, and repeatedly centrifuging for several times.
8. The separation and purification method according to claim 1, wherein in the step 4), the density gradient separation liquid has a density difference of 1.06-1.10g/cm3With a continuous density gradient separation liquid in between.
9. The separation and purification method according to claim 8, wherein the preparing the continuous density gradient separation liquid comprises the following raw materials: the density was 1.06g/cm3Has a low density gradient separation liquid and a density of 1.10g/cm3High density gradient separation of liquids.
10. The separation and purification method according to claim 1, wherein in the step 2), the size of the pancreatic block is a small block with a side length of about 2 cm.
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