CN111909896B - Method for extracting adipose tissue stem cells - Google Patents

Method for extracting adipose tissue stem cells Download PDF

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CN111909896B
CN111909896B CN202010788902.1A CN202010788902A CN111909896B CN 111909896 B CN111909896 B CN 111909896B CN 202010788902 A CN202010788902 A CN 202010788902A CN 111909896 B CN111909896 B CN 111909896B
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bag
cleaning
digestion
adipose tissue
outlet
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CN111909896A (en
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李伟强
胡旺
邹桥
刘亚琼
赵乾润
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Zhongke Kangsai Biotechnology Chongqing Co ltd
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Zhongke Kangsai Biotechnology Chongqing Co ltd
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Abstract

The invention discloses an extraction method of adipose tissue stem cells, which adopts a multi-bag adipose tissue stem cell extraction device for extraction and comprises the following steps: (A1) 30mL of adipose tissue active agent is added in the first cleaning bag in advance; (A2) adding the adipose tissues into the first cleaning bag to uniformly mix the adipose tissues with the adipose tissue active agent; (A3) centrifuging the first cleaning bag; (A4) centrifuging the third cleaning bag; (A5) transferring into a digestion bag; (A6) - (A8) digesting in a digestion bag, and centrifuging. The adipose tissue stem cell extraction method provided by the invention provides a relatively closed sterile internal environment for transportation, cleaning and digestion of adipose tissues, reduces the chance of contact with the outside, reduces the probability of adipose tissue pollution, ensures the safety of experiments, and further improves the efficiency of adipose tissue cleaning and digestion.

Description

Method for extracting adipose tissue stem cells
Technical Field
The invention relates to the field of adipose tissue stem cells, in particular to an extraction method of adipose tissue stem cells.
Background
Adipose tissue is abundant in human body, a large number of adipose-derived stem cells (ADSCs) obtained by liposuction have the potential of self-renewal, proliferation and multidirectional differentiation, can be differentiated into adipocytes, chondrocytes, myocytes, osteoblasts, nerve cells, glial cells and islet cells, can secrete various angiogenesis promoting factors and anti-apoptosis factors to resist inflammation and oxidation, can resist the damage of oxygen free radicals, and is expected to become a stem cell source for repairing damaged tissues and organs.
At present, most of the collection and transportation of adipose tissues directly select an injector used in liposuction as a carrier, so that the problems of leakage and pollution are very easy to occur in the transportation process, and long-time transportation and uncontrolled external environment are very easy to influence the activity of adipose stem cells. Adipose tissue transports to the laboratory after, and the centrifuging tube that adopts more in the extraction process is as the container, and is repeated to it wash and centrifugation, uses erlenmeyer flask or centrifuging tube to digest adipose tissue as the container after the washing is accomplished, and this washing and digestion process are open application of sample and sample, very easily cause the pollution.
In addition, carry out fat digestion with traditional three-dimensional container and can not all immerse in the water to thermally equivalent in the container can't guaranteeing all the time, again traditional container thermal conductivity is not too good and be difficult for observing, thereby leads to adipose tissue digestion time long, and the inhomogeneous condition of digestion appears.
Disclosure of Invention
The invention provides an extraction method of adipose tissue stem cells, aiming at the problem that the existing adipose tissues are easy to leak and pollute in the transportation, cleaning and digestion processes, and the extraction method ensures that the adipose tissues are in a relatively closed sterile internal environment in the transportation, cleaning and digestion processes, thereby reducing the probability of adipose tissue pollution.
In order to achieve the purpose, the technical scheme of the invention is as follows: a method for extracting adipose tissue stem cells adopts a multi-bag adipose tissue stem cell extraction device for extraction, the multi-bag adipose tissue stem cell extraction device comprises a first cleaning bag, a second cleaning bag, a third cleaning bag and a digestion bag which are sequentially connected, and the first cleaning bag, the second cleaning bag, the third cleaning bag and the digestion bag are mutually communicated through diversion hoses; the upper part of the bag body of the digestion bag is provided with two sample inlets and outlets, one sample inlet and outlet is digestive juice, and the other sample inlet and outlet is integrally in a branch shape and comprises a main sample inlet and outlet and two branch sample inlets and outlets;
the method comprises the following steps:
(A1) 30mL of adipose tissue active agent is pre-added into the first cleaning bag (1);
(A2) closing a flow guide hose between the first cleaning bag and the second cleaning bag, extracting 50-80mL of adipose tissues in an aseptic environment, injecting the adipose tissues into the first cleaning bag through the first sample inlet and outlet, and shaking the first cleaning bag to uniformly mix the adipose tissues and the adipose tissue active agent;
(A3) injecting a cleaning solution with the same volume as the adipose tissues from the second sample inlet and outlet, and shaking the first cleaning bag to fully and uniformly mix the adipose tissues and the cleaning solution;
(A4) injecting a cleaning solution with the same volume as the adipose tissues from the third sample inlet and outlet, and shaking the second cleaning bag to fully and uniformly mix the adipose tissues and the cleaning solution; centrifuging the second cleaning bag at the rotating speed of 400-600 rpm for 5-8min, and allowing upper-layer adipose tissues and a small amount of grease to flow into a third cleaning bag through a diversion hose;
(A5) injecting a cleaning solution with the same volume as the adipose tissues from the fourth sample inlet and outlet, and shaking the third cleaning bag to fully and uniformly mix the adipose tissues and the cleaning solution; centrifuging the third cleaning bag, wherein the centrifugal rotating speed is 400-600 rpm, and the centrifuging time is 5-8 min; after cleaning and centrifugation, transferring the adipose tissues into the digestion bag;
(A6) injecting collagenase I digestive juice with the same volume as the fat tissue in the digestive bag from a digestive juice inlet and outlet, then immersing the digestive bag in water bath at 37 ℃, fixing the digestive bag on a bottom plate of a water bath shaker for vibration digestion through magnetic force, carrying out vibration digestion for 30-40min at the rotation speed of 100-200rpm, and observing the digestion condition of the fat tissue at regular time;
(A7) after digestion is finished, injecting a mesenchymal stem cell serum-free complete culture medium with the volume equal to that of liquid in the digestion bag through a branch sample inlet and outlet on the digestion bag to terminate digestion, and after uniform mixing, carrying out centrifugal treatment at the centrifugal rotation speed of 1400-1600rpm for 5-8 min;
(A8) and after the centrifugation is finished, pumping the upper layer grease, the undigested complete adipose tissues and the redundant solution from the other branch sample inlet and outlet of the digestion bag, and pumping the residual single cell suspension from the main sample inlet and outlet, thus finishing the extraction of the adipose tissue stem cells.
According to the method for extracting the adipose tissue stem cells, the three cleaning bags and the one digestion bag are arranged, and the whole device keeps a relatively closed sterile environment, so that the cleaning and the digestion of the adipose tissue stem cells can be carried out in the closed environment, and the probability of adipose tissue pollution is reduced; in addition, the adipose tissue active agent is added in the first cleaning bag in advance, so that the adipose tissues collected in the process of being injected into the first cleaning bag for long-time transportation can protect the activity of the adipose tissues, improve the activity of adipose stem cells, collect and precipitate red blood cells mixed in the adipose tissues, and optimize the cleaning efficiency.
Further, the adipose tissue active agent comprises the following components in percentage by mass: 5500.5-1% of hydroxyethyl starch, 02-0.4% of trehalose, 0.8-1.2% of human serum albumin, 0.1% of tribasic sodium citrate and the balance of PBS buffer solution. So as to protect the activity of the adipose tissues during long-term transportation.
Furthermore, every go into out the appearance mouth through the water conservancy diversion hose with the internal space of the bag body of first washing bag, second washing bag, third washing bag and digestion bag is connected. The sample inlet and outlet can be thermally sealed with the diversion hose close to the end of the bag body after sample adding or sample discharging is completed, and then the used part is removed, so that the bag body is completely isolated from the outside during water bath digestion, and the bag body is prevented from being polluted.
Further, the bag body of digestion bag is the fillet rectangle be equipped with a plurality of strong magnet subsides and attaches the point on the bag body, the bag body passes through strong magnet pastes the point and sets up strong magnet piece to make through the magnetic force of strong magnet piece and constant temperature water bath shaking table the bag body is fixed on the constant temperature water bath shaking table. A plurality of strong magnet pastes the point setting at four angles of the bag body, and the digestion bag is through setting up strong magnet adsorption site on the bag body like this, utilizes the strong magnet piece with the bag body snap-on the constant temperature water bath shaking table, need not to fix with the help of other instruments, just can be so that whole bag body submergence is in constant temperature aquatic for digestion efficiency, improved adipose stem cell's separation extraction efficiency and quality greatly, solved the problem that traditional three-dimensional container can not all submergence aquatic.
Furthermore, a filtering device is arranged on the main sample inlet and outlet. Therefore, when the adipose-derived stem cell suspension is finally extracted, fine adipose tissues can be filtered out, so that the obtained cell suspension can be directly subjected to subsequent operation without additional filtering operation. In addition, this filter equipment is double-deck filter screen structure, and wherein the aperture that is close to the one deck filter screen of the bag body is 300um, and the aperture of another layer filter screen is 100um, and this design can effectively prevent the shutoff when filtering.
Furthermore, moulding metal strips are embedded in the edges of the two sides of the bag body of the first cleaning bag, the second cleaning bag, the third cleaning bag and the digestion bag respectively. This moulding metal strip has certain rigidity intensity, and can take place deformation again, both can prevent like this that the bag body from taking place deformation fold and piling up because of centrifugal force when the bag body is centrifugal, can artificially buckle the metal strip again and drive the bag body and do certain deformation.
Furthermore, each end part of the sample inlet and outlet is provided with a twist-off sealing rubber ring, and each internal interface of the sample inlet and outlet is matched with the nipple interface of the injector. All the sample inlets and outlets are completely sealed in advance, the front end part needs to be twisted off when the sample inlet and outlet bag is used, the sample inlet and outlet operation can be carried out only by exposing the internal interfaces of the sample inlets and outlets, and the inner part of the bag body is in a completely sealed state through the diversion hose connected by heat sealing after the sample inlet and outlet bag is used, so that the risk of pollution is further reduced.
Furthermore, all the diversion hoses of the sample inlet and outlet are sealed in a heat seal mode after being used. This allows the entire extraction device to be in a completely closed state, avoiding contamination of the adipose tissue in the bag.
Furthermore, scales are arranged on the surfaces of the first cleaning bag, the second cleaning bag, the third cleaning bag and the digestion bag. Therefore, when the sample feeding and discharging operation is carried out, the judgment of the sample feeding and discharging volume by an operator can be well assisted, and the controllability and the accuracy of the experiment are facilitated.
Furthermore, the diversion hose and the bag body are made of transparent PVC with high resistance, high toughness, heat sealability and strong hydrophobicity and oleophobicity. Therefore, after the adipose tissues are centrifugally layered, the components are clamped and separated layer by layer more smoothly; during water bath, the temperature conduction is faster, and the digestion time of tissues can be shortened; meanwhile, the transparent bag body is convenient for the operation and observation of the whole experimental process. In addition, the diversion hose can be sealed through heat sealing after each operation, used parts are omitted, the encumbrance of redundant parts is reduced, and the sealing performance of the environment in the bag body is guaranteed.
Has the advantages that:
the adipose tissue stem cell extraction method adopts the multi-bag adipose tissue stem cell extraction device for extraction, provides a relatively closed sterile internal environment for transportation, cleaning and digestion of adipose tissues, reduces the chance of contact with the outside, reduces the probability of adipose tissue pollution, and ensures the safety of experiments; meanwhile, the adipose tissue active agent in the first cleaning bag can play a role in maintaining the adipose tissue activity in the long-time transportation process, so that the adipose tissue cleaning efficiency is further improved; in addition, the bag type digestion mode increases the water bath area, greatly shortens the water bath digestion time and improves the digestion efficiency.
Drawings
FIG. 1 is a schematic view of an apparatus used in the method for extracting adipose tissue stem cells of the present invention;
FIG. 2 is a partial schematic view of a sample inlet and outlet;
FIG. 3 is a schematic view of a filtration apparatus;
FIG. 4 is a schematic view of a liquid stop clip;
FIG. 5 is a graph showing the assay results of example 1;
FIG. 6 is a photograph showing the direct centrifugation effect of the collected adipose tissues in the bag;
FIG. 7 is a graph showing the results of digestion of adipose tissue in the digestion bag for 35min and centrifugation in example 1;
FIG. 8 is a photograph of the layer-by-layer separation and transfer of components using a blood plasma clamp after digestion of adipose tissue.
Labeled as: 1 a first cleaning bag, 2 a second cleaning bag, 3 a third cleaning bag, 4 a digestion bag, 5 a diversion hose, 6 an adipose tissue active agent, 7 a strong magnet attachment point, 8 a digestive juice sample inlet and outlet port, 9a main sample inlet and outlet port, 10 a branch sample inlet and outlet port, 11 a filtering device, 12 a shaping metal strip, 13 a twist-off sealing rubber ring, 14 an internal interface, 15 scales, 16 a first sample inlet and outlet port, 17 a second sample inlet and outlet port, 18 a third sample inlet and outlet port, 19 a fourth sample inlet and outlet port and 20 a liquid stopping clamp.
Detailed Description
An adipose tissue stem cell extraction method adopts a multi-bag adipose tissue stem cell extraction device for extraction, the multi-bag adipose tissue stem cell extraction device comprises a first cleaning bag 1, a second cleaning bag 2, a third cleaning bag 3 and a digestion bag 4 which are sequentially connected, and the first cleaning bag 1, the second cleaning bag 2, the third cleaning bag 3 and the digestion bag 4 are mutually communicated through a diversion hose 5; the upper part of the bag body of the digestion bag 4 is provided with two sample inlets and outlets, one sample inlet and outlet is a digestive juice sample inlet and outlet 8, and the other sample inlet and outlet is integrally in a branch shape and comprises a main sample inlet and outlet 9 and two branch sample inlets and outlets 10;
the method comprises the following steps:
(A1) 30mL of adipose tissue active agent 6 is added in the first cleaning bag 1 in advance;
(A2) closing the diversion hose 5 between the first cleaning bag 1 and the second cleaning bag 2, extracting 50-80mL of adipose tissues in a sterile environment, injecting the adipose tissues into the first cleaning bag 1 through the first sample inlet and outlet 16, and shaking the first cleaning bag 1 to uniformly mix the adipose tissues and the adipose tissue active agent;
(A3) injecting a cleaning solution with the same volume as the adipose tissues from the second sample inlet and outlet 17, and shaking the first cleaning bag 1 to fully and uniformly mix the adipose tissues and the cleaning solution; centrifuging the first cleaning bag 1 at the rotating speed of 400-600 rpm for 5-8min, and making the upper layer adipose tissues and a small amount of grease flow into the second cleaning bag 2 through a diversion hose 5;
(A4) injecting a cleaning solution with the same volume as the adipose tissues from the third sample inlet and outlet 18, and shaking the second cleaning bag 2 to fully and uniformly mix the adipose tissues and the cleaning solution; centrifuging the second cleaning bag 2 at the rotating speed of 400-600 rpm for 5-8min, and making the upper layer adipose tissues and a small amount of grease flow into a third cleaning bag 3 through a diversion hose 5;
(A5) injecting a cleaning solution with the same volume as the adipose tissues from the fourth sample inlet and outlet 19, and shaking the third cleaning bag 3 to fully and uniformly mix the adipose tissues and the cleaning solution; centrifuging the third cleaning bag 3 at the rotating speed of 400-600 rpm for 5-8 min; after cleaning and centrifugation, transferring the adipose tissues into the digestion bag 4;
(A6) injecting I-type collagenase digestion solution with the same volume as the fat tissue in the digestion bag 4 from a digestion solution inlet and outlet 8, then immersing the digestion bag 4 in water bath at 37 ℃, fixing the digestion bag 4 on a bottom plate of a water bath shaker through magnetic force for vibration digestion, carrying out vibration digestion for 30-40min at the rotation speed of 100-200rpm, and observing the digestion condition of the fat tissue at regular time;
(A7) after digestion is finished, injecting a mesenchymal stem cell serum-free complete culture medium with the volume equal to that of liquid in the digestion bag through a branch sample inlet and outlet 10 on the digestion bag 4 to terminate digestion, and after uniform mixing, carrying out centrifugal treatment at the centrifugal rotation speed of 1400-1600rpm for 5-8 min;
(A8) after the centrifugation is finished, the upper layer grease, the undigested complete adipose tissues and the redundant solution are extracted from the other branch sample inlet and outlet 10 of the digestion bag 4, and the residual single cell suspension is extracted from the main sample inlet and outlet 9, thus finishing the extraction of the adipose tissue stem cells.
Wherein the adipose tissue active agent 6 comprises the following components in percentage by mass: 5500.5-1% of hydroxyethyl starch, 02-0.4% of trehalose, 0.8-1.2% of human serum albumin, 0.1% of tribasic sodium citrate and the balance of PBS buffer solution.
Wherein, every the business turn over appearance mouth through water conservancy diversion hose 5 with the internal space of the bag of first washing bag 1, second washing bag 2, third washing bag 3 and digestion bag 4 is connected.
The digestion bag 4 is characterized in that the bag body is a rounded rectangle, a plurality of strong magnet attachment points 7 are arranged on the bag body, the bag body passes through the strong magnet attachment points 7 and is provided with a strong magnet block, and the bag body is fixed on the constant-temperature water bath rocking bed through the strong magnet block and the magnetic force of the constant-temperature water bath rocking bed.
Wherein, a filter device 11 is arranged on the main sample inlet and outlet 9.
Wherein, the two side edges of the first cleaning bag 1, the second cleaning bag 2, the third cleaning bag 3 and the digestion bag 4 are respectively embedded with a shaping metal strip 12.
Wherein, but every the tip of business turn over appearance mouth all is equipped with twist-off formula sealing rubber ring 13, and every the inside interface 14 of business turn over appearance mouth and the nipple interface looks adaptation of syringe.
And after all the diversion hoses of the sample inlet and outlet are used, the diversion hoses are sealed in a heat sealing mode.
Scales 15 are arranged on the surfaces of the first cleaning bag 1, the second cleaning bag 2, the third cleaning bag 3 and the digestion bag 4.
The diversion hose 5 and the bag body are made of transparent PVC with high resistance, high toughness, heat sealability and strong hydrophobicity and oleophobicity.
The internal shapes of the three cleaning bags are all rectangles with the upper ends protruding outwards and shrinking, so that fat is conveniently cleaned and centrifuged in the bags, and fat tissues are transferred from the diversion hose through clamping; and the internal shape of the digestion bag body is circular, which is beneficial to the regular flow of the internal solution of the digestion bag body when the digestion bag body is vibrated in a water bath shaker, and the uniformity of the digestion degree of the adipose tissues is ensured.
The opening and closing of all the diversion hoses in the invention can be controlled by the liquid stopping clamp 20.
The present invention will be further described with reference to the following specific examples.
Example 1
An adipose tissue stem cell extraction method adopts a multi-bag adipose tissue stem cell extraction device for extraction, and comprises the following steps:
(A) extraction of adipose tissue stem cells:
(A1) 30mL of adipose tissue active agent 6 is added in the first cleaning bag 1 in advance;
(A2) clamping and sealing the diversion hose 5 between the first cleaning bag 1 and the second cleaning bag 2 by using a liquid stopping clamp 20, extracting 50mL of adipose tissues in a sterile environment, injecting the adipose tissues into the first cleaning bag 1 through the first sample inlet and outlet 16, and then thermally sealing the diversion hose 5 of the first sample inlet and outlet 16 to ensure that the interior of the first cleaning bag 1 is in a sealed state; shaking the first cleaning bag 1 to uniformly mix the adipose tissues and the adipose tissue active agent, so as to ensure that the activity of the adipose tissues is protected in the long-time transportation process;
(A3) injecting a 50mLPBS cleaning solution from the second sample inlet and outlet 17, then sealing the guide hose 5 of the second sample inlet and outlet 17 in a heat sealing manner, and shaking the first cleaning bag 1 to fully and uniformly mix the adipose tissues and the cleaning solution;
(A4) laminating the four bag bodies together, placing the bag bodies into a centrifugal adapter for vertical fixation, and centrifuging at the rotation speed of 400rpm for 8 min; stably taking out the multi-connected bag body after centrifugation is finished, clamping the first cleaning bag 1 by a blood plasma separating clamp, clamping and sealing a diversion hose 5 between the second cleaning bag 2 and the third cleaning bag 3 by a liquid stopping clamp 20, removing the liquid stopping clamp 20 between the first cleaning bag 1 and the second cleaning bag 2, slowly flowing upper layer adipose tissues and a small amount of grease into the second cleaning bag 2 through the diversion hose 5 by controlling the blood plasma separating clamp, remaining lower layer transparent solution and red cell sediments in the first cleaning bag 1, thermally sealing the diversion hose 5 between the first cleaning bag 1 and the second cleaning bag 2, and reducing the first cleaning bag 1;
(A5) repeating the steps (A3) and (a4), performing second and third washing centrifugation on the adipose tissues in the second washing bag 2 and the third washing bag 3, and transferring the adipose tissues into the digestion bag 4 after the washing centrifugation; sealing the diversion hose 5 between the third cleaning bag 3 and the digestion bag 4 by heat seal, and removing the third cleaning bag 3;
(A6) injecting I type collagenase digestion solution with the same volume as the fat tissue in the digestion bag 4 from a digestion solution inlet and outlet sample port 8, then thermally sealing a diversion hose 5 of the digestion solution inlet and outlet sample port 8, immersing the digestion bag 4 in a water bath at 37 ℃, fixing the digestion bag on a bottom plate of a water bath shaker for vibration digestion under the action of magnetic force, carrying out vibration digestion at the rotating speed of 100rpm for 30min, and regularly observing the digestion condition of the fat tissue;
(A7) after digestion is finished, injecting a mesenchymal stem cell serum-free complete culture medium with the volume equal to that of liquid in the digestion bag through a branch sample inlet and outlet 10 on the digestion bag 4 to terminate digestion, then sealing a diversion hose 5 of the branch sample inlet and outlet 10 in a heat sealing manner, uniformly mixing, and then carrying out centrifugal treatment at a centrifugal rotating speed of 1400rpm for 8 min;
(A8) after centrifugation is finished, a grease layer, a part of undigested fat tissue layer, a transparent solution layer and a cell precipitation layer are respectively arranged in the digestion bag 4 from top to bottom, the digestion bag is clamped by a blood plasma separating clamp to extract upper grease, undigested complete fat tissue and redundant solution from the sample inlet and outlet 10 of the other branch of the digestion bag 4, the diversion hose 5 of the sample inlet and outlet 10 of the other branch is sealed by a liquid stopping clamp 20, and after the residual solution and precipitated cells in the digestion bag 4 are uniformly mixed, the residual solution and the precipitated cells are extracted from the main sample inlet and outlet 9 through a filter device 11, so that the extraction of the fat tissue dry cells is finished;
(B) culture of adipose tissue stem cells
(B1) The single cell suspension obtained above was resuspended to 50ml with PBS and centrifuged at 1400rpm for 8 min.
(B2) Discarding the supernatant, adding 30mL of culture medium into the lower layer solution to resuspend the cell liquid, inoculating into 2T 75 culture bottles, culturing in a 5% carbon dioxide incubator at 37 ℃ for 12-24 h;
(B3) liquid changing: collecting supernatant in a cell culture bottle, centrifuging, adding a mesenchymal stem cell serum-free complete culture medium for resuspension, and adding the mixture into the original culture bottle for continuous culture;
(B4) passage: replacing the culture medium every 3 days, when the cell fusion degree reaches 80%, cleaning with normal saline for 2 times, adding pancreatin for digestion for 4-8min, adding normal saline or mesenchymal stem cell serum-free complete culture medium to terminate digestion, and harvesting the adipose tissue stem cell.
Example 2
An adipose tissue stem cell extraction method adopts a multi-bag adipose tissue stem cell extraction device for extraction, and comprises the following steps:
(A) extraction of adipose tissue stem cells:
(A1) 30mL of adipose tissue active agent 6 is added in the first cleaning bag 1 in advance;
(A2) clamping and sealing the diversion hose 5 between the first cleaning bag 1 and the second cleaning bag 2 by using a liquid stopping clamp 20, extracting 75mL of adipose tissues in a sterile environment, injecting the adipose tissues into the first cleaning bag 1 through the first sample inlet and outlet 16, and then thermally sealing the diversion hose 5 of the first sample inlet and outlet 16 to ensure that the interior of the first cleaning bag 1 is in a sealed state; shaking the first cleaning bag 1 to uniformly mix the adipose tissues and the adipose tissue active agent, so as to ensure that the activity of the adipose tissues is protected in the long-time transportation process;
(A3) injecting 75mL PBS cleaning fluid from the second sample inlet and outlet 17, then sealing the guide hose 5 of the second sample inlet and outlet 17 in a heat sealing way, and shaking the first cleaning bag 1 to fully and uniformly mix the adipose tissues and the cleaning fluid;
(A4) laminating the four bag bodies together, placing the bag bodies into a centrifugal adapter for vertical fixation, and centrifuging at the centrifugal rotation speed of 500rpm for 7 min; stably taking out the multi-connected bag body after centrifugation is finished, clamping the first cleaning bag 1 by a blood plasma separating clamp, clamping and sealing a diversion hose 5 between the second cleaning bag 2 and the third cleaning bag 3 by a liquid stopping clamp 20, removing the liquid stopping clamp 20 between the first cleaning bag 1 and the second cleaning bag 2, slowly flowing upper layer adipose tissues and a small amount of grease into the second cleaning bag 2 through the diversion hose 5 by controlling the blood plasma separating clamp, remaining lower layer transparent solution and red cell sediments in the first cleaning bag 1, thermally sealing the diversion hose 5 between the first cleaning bag and the second cleaning bag 2 of 1, and reducing the first cleaning bag 1;
(A5) repeating the steps (A3) and (a4), performing second and third washing centrifugation on the adipose tissues in the second washing bag 2 and the third washing bag 3, and transferring the adipose tissues into the digestion bag 4 after the washing centrifugation; sealing the diversion hose 5 between the third cleaning bag 3 and the digestion bag 4 by heat seal, and removing the third cleaning bag 3;
(A6) injecting I type collagenase digestion solution with the same volume as the fat tissue in the digestion bag 4 from a digestion solution inlet and outlet sample port 8, then thermally sealing a diversion hose 5 of the digestion solution inlet and outlet sample port 8, immersing the digestion bag 4 in a water bath at 37 ℃, fixing the digestion bag on a bottom plate of a water bath shaker for vibration digestion under the action of magnetic force, carrying out vibration digestion at the rotating speed of 150rpm for 35min, and regularly observing the digestion condition of the fat tissue;
(A7) after digestion is finished, injecting a mesenchymal stem cell serum-free complete culture medium through a branch sample inlet and outlet 10 on the digestion bag 4, then thermally sealing a diversion hose 5 of the branch sample inlet and outlet 10, uniformly mixing, and then carrying out centrifugal treatment at a centrifugal rotating speed of 1500rpm for 7 min;
(A8) after centrifugation is finished, a grease layer, a part of undigested fat tissue layer, a transparent solution layer and a cell precipitation layer are respectively arranged in the digestion bag 4 from top to bottom, the digestion bag is clamped by a blood plasma separating clamp to extract upper grease, undigested complete fat tissue and redundant solution from the sample inlet and outlet 10 of the other branch of the digestion bag 4, a diversion hose 5 of the sample inlet and outlet 10 of the other branch is sealed by a liquid stopping clamp 20, and after the residual solution and precipitated cells in the digestion bag are uniformly mixed, the residual solution and the precipitated cells are extracted from a main sample inlet and outlet 9 through a filter device 11, so that the extraction of the fat tissue stem cells is finished;
(B) culture of adipose tissue stem cells
(B1) The single cell suspension obtained above was resuspended to 50mL with PBS and centrifuged at 1300rpm for 6 min.
(B2) Discarding the supernatant, adding 30mL of culture medium into the supernatant to resuspend the cell liquid, inoculating into 2T 75 culture bottles, culturing at 37 deg.C in a 5% carbon dioxide incubator for 12-24 h;
(B3) liquid changing: collecting supernatant in a cell culture bottle, centrifuging, adding a mesenchymal stem cell serum-free complete culture medium for resuspension, and adding the mixture into the original culture bottle for continuous culture;
(B4) passage: replacing the culture medium every 3 days, when the cell fusion degree reaches 80%, cleaning with normal saline for 2 times, adding pancreatin for digestion for 4-8min, adding normal saline or mesenchymal stem cell serum-free complete culture medium to terminate digestion, and harvesting the adipose tissue stem cell.
Example 3
An adipose tissue stem cell extraction method adopts a multi-bag adipose tissue stem cell extraction device for extraction, and comprises the following steps:
(A) extraction of adipose tissue stem cells:
(A1) 30mL of adipose tissue active agent 6 is added in the first cleaning bag 1 in advance;
(A2) clamping and sealing the diversion hose 5 between the first cleaning bag 1 and the second cleaning bag 2 by using a liquid stopping clamp 20, extracting 80mL of adipose tissues in an aseptic environment, injecting the adipose tissues into the first cleaning bag 1 through the first sample inlet and outlet 16, and then thermally sealing the diversion hose 5 of the first sample inlet and outlet 16 to ensure that the interior of the first cleaning bag 1 is in a sealed state; shaking the first cleaning bag 1 to uniformly mix the adipose tissues and the adipose tissue active agent, so as to ensure that the activity of the adipose tissues is protected in the long-time transportation process;
(A3) injecting 80mLPBS cleaning solution from the second sample inlet and outlet 17, then sealing the guide hose 5 of the second sample inlet and outlet 17 in a heat sealing manner, and shaking the first cleaning bag 1 to fully and uniformly mix the adipose tissues and the cleaning solution;
(A4) laminating the four bag bodies together, placing the bag bodies into a centrifugal adapter for vertical fixation, and centrifuging at the rotating speed of 600rpm for 5 min; after centrifugation, the multi-connected bag body is stably taken out, the first cleaning bag 1 is clamped by a blood plasma separating clamp, the diversion soft pipe 5 between the second cleaning bag 2 and the third cleaning bag 3 is clamped and sealed by a liquid stopping clamp 20, the liquid stopping clamp 20 between the first cleaning bag 1 and the second cleaning bag 2 is removed, upper layer adipose tissues and a small amount of grease flow into the second cleaning bag 2 through the diversion hose 5 by controlling the blood plasma separating clamp slowly, lower layer transparent solution and red cell sediments are remained in the first cleaning bag 1, the diversion hose 5 between the first cleaning bag 1 and the second cleaning bag 2 is sealed in a heat sealing mode, and the first cleaning bag 1 is removed;
(A5) repeating the steps (A3) and (a4), performing second and third washing centrifugation on the adipose tissues in the second washing bag 2 and the third washing bag 3, and transferring the adipose tissues into the digestion bag 4 after the washing centrifugation; sealing the diversion hose 5 between the third cleaning bag 3 and the digestion bag 4 by heat seal, and removing the third cleaning bag 3;
(A6) injecting I type collagenase digestion solution with the same volume as the fat tissue in the digestion bag 4 from a digestion solution inlet and outlet sample port 8, then thermally sealing a diversion hose 5 of the digestion solution inlet and outlet sample port 8, immersing the digestion bag 4 in a water bath at 37 ℃, fixing the digestion bag on a bottom plate of a water bath shaker for vibration digestion under the action of magnetic force, carrying out vibration digestion at the rotation speed of 200rpm for 40min, and regularly observing the digestion condition of the fat tissue;
(A7) after digestion is finished, injecting a mesenchymal stem cell serum-free complete culture medium with the volume equal to that of liquid in the digestion bag through a branch sample inlet and outlet 10 on the digestion bag 4 to terminate digestion, then sealing a diversion hose 5 of the branch sample inlet and outlet 10 in a heat sealing manner, uniformly mixing, and then carrying out centrifugal treatment at a centrifugal rotating speed of 1600rpm for 5 min;
(A8) after centrifugation is finished, a grease layer, a part of undigested fat tissue layer, a transparent solution layer and a cell precipitation layer are respectively arranged in the digestion bag 4 from top to bottom, the digestion bag is clamped by a blood plasma separating clamp to extract upper grease, undigested complete fat tissue and redundant solution from the sample inlet and outlet 10 of the other branch of the digestion bag 4, a diversion hose 5 of the sample inlet and outlet 10 of the other branch is sealed by a liquid stopping clamp 20, and after the residual solution and precipitated cells in the digestion bag are uniformly mixed, the residual solution and the precipitated cells are extracted from a main sample inlet and outlet 9 through a filter device 11, so that the extraction of the fat tissue stem cells is finished;
(B) culture of adipose tissue stem cells
(B1) The single cell suspension obtained above was resuspended to 50mL with PBS and centrifuged at 1300rpm for 6 min.
(B2) Discarding the supernatant, adding 30mL of culture medium into the supernatant to resuspend the cell liquid, inoculating into 2T 75 culture bottles, culturing at 37 deg.C in a 5% carbon dioxide incubator for 12-24 h;
(B3) liquid changing: collecting supernatant in a cell culture bottle, centrifuging, adding a mesenchymal stem cell serum-free complete culture medium for resuspension, and adding the mixture into the original culture bottle for continuous culture;
(B4) passage: replacing the culture medium every 3 days, when the cell fusion degree reaches 80%, cleaning with normal saline for 2 times, adding pancreatin for digestion for 4-8min, adding normal saline or mesenchymal stem cell serum-free complete culture medium to terminate digestion, and harvesting the adipose tissue stem cell.
In examples 1 to 3, a replanting step may be added after step (B4), specifically: and (4) washing the culture bottle digested in the step (B4) with PBS for 2 times, adding a mesenchymal stem cell serum-free complete culture medium into the adipose tissue stem cells which are not completely digested, continuing to culture, repeating the step (B4), and finally harvesting the adipose tissue stem cells.
In examples 1 to 3, the added adipose tissue-derived agent 6 comprises the following components in percentage by mass: 5500.5-1% of hydroxyethyl starch, 02-0.4% of trehalose, 0.8-1.2% of human serum albumin, 0.1% of tribasic sodium citrate and the balance of PBS buffer solution.
Comparative example 1
The traditional method for extracting adipose-derived stem cells by using a centrifugal tube method comprises the following steps:
(1) adding 50mL of aseptically obtained adipose tissue into a centrifuge tube, resuspending the adipose tissue by using physiological saline containing three antibiotics with equal volume, centrifuging for 6min in a centrifuge at the rotating speed of 600rpm, removing lower-layer liquid by using a pipette, adding the physiological saline with equal volume again, shaking and mixing uniformly, centrifuging for 6min at the rotating speed of 600rpm until the physiological saline is transparent, and removing blood cells and other impurities;
(2) adding the homogenized adipose tissue into type I collagenase with the same volume, placing the mixture into a constant-temperature water bath shaking box, digesting the mixture for 30min in a water bath at 37 ℃ and at 150rpm, adding 20mL of complete serum-free culture medium to stop digestion, and centrifuging the mixture for 8min at 1500 rpm;
(3) and (3) adding 2 times of volume of physiological saline into the fat cells obtained in the step (2) for resuspension, centrifuging at the rotating speed of 1500rpm for 8min, and discarding the supernatant solution.
(4) Filtering the adipose tissue and cell homogenate after the normal saline is resuspended and centrifuged, centrifuging for 8min at the rotating speed of 1500rpm, and discarding the upper solution to obtain the adipose-derived stem cells.
Comparative example 2
The traditional method for extracting adipose-derived stem cells by using a centrifugal tube method comprises the following steps:
(1) adding 75mL of aseptically obtained adipose tissue into a centrifuge tube, resuspending the adipose tissue by using physiological saline containing three antibiotics with equal volume, centrifuging for 7min at the rotating speed of 500rpm in a centrifuge, removing lower-layer liquid by using a pipette, adding the physiological saline with equal volume again, shaking and mixing uniformly, centrifuging for 7min at the rotating speed of 500rpm until the physiological saline is transparent, and removing blood cells and other impurities;
(2) adding the homogenized adipose tissue into type I collagenase with the same volume, placing the mixture in a constant-temperature water bath shaking box, digesting the mixture for 25min in a water bath at 38 ℃ and at 200rpm, adding 20mL of complete serum-free culture medium to stop digestion, and centrifuging the mixture for 6min at 1800 rpm;
(3) adding 2 times volume of physiological saline into the fat cells obtained in the step (2) for resuspension, centrifuging at 1800rpm for 6min, and removing supernatant solution;
(4) resuspending centrifuged adipose tissues and cell homogenate by normal saline, filtering, centrifuging at 1800rpm for 6min, and discarding the supernatant solution to obtain adipose stem cells.
Comparative example 3
The traditional method for extracting adipose-derived stem cells by using a centrifugal tube method comprises the following steps:
(1) adding 80mL of aseptically obtained adipose tissue into a centrifuge tube, resuspending the adipose tissue by using physiological saline containing three antibiotics with equal volume, centrifuging for 8min at the rotating speed of 400rpm in a centrifuge, removing lower-layer liquid by using a pipette, adding the physiological saline with equal volume again, shaking and uniformly mixing, centrifuging for 8min at the rotating speed of 400rpm until the physiological saline is transparent, and removing blood cells and other impurities;
(2) adding the homogenized adipose tissue into type I collagenase of the same volume, placing in a constant temperature water bath shaking box, digesting for 35min in water bath at 36 ℃ and 100rpm, adding 20ml of complete serum-free culture medium to stop digestion, and centrifuging for 7min at 1600 rpm;
(3) adding 2 times of volume of physiological saline into the fat cells obtained in the step (2) for resuspension, centrifuging for 7min at the rotating speed of 1600rpm, and discarding supernatant solution;
(4) resuspending centrifuged adipose tissues and cell homogenate by normal saline, filtering, centrifuging at 1600rpm for 7min, and discarding the supernatant solution to obtain adipose stem cells.
Table 1 shows the reagents used in the culture of adipose tissue stem cells according to the present invention
Figure BDA0002623045570000151
Table 2 shows the apparatus used in the adipose tissue stem cell culture process of the present invention
Serial number Name (R) Model number Manufacturer of the product Status of state
1 Biological safety cabinet 1200C Li Shen Has been verified
2 Carbon dioxide incubator 3111 Thermo Has been verified
3 Constant-temperature water bath oscillation box Jinghong tablet Has been verified
4 Centrifugal machine ST16 Thermo Has been verified
5 Flow cytometer C6PLUS BD Has been verified
6 Blood plasma separating clamp Fixing device
7 Floor type centrifuge 6000i Thermo Has been verified
Table 3 shows the results of examples 1 to 3 in comparison with comparative examples 1 to 3
Figure BDA0002623045570000161
As can be seen from the above table, in examples 1 to 3, in comparison with comparative examples 1 to 3, in the adipose tissue stem cell culture method provided by the present invention, the time for complete digestion of adipose tissue stem cells with the same volume is significantly shorter than that in the comparative examples, and it can be seen that the efficiency of adipose tissue digestion is improved by using the digestion method of the multiple connected bags of adipose tissue stem cells provided by the present invention; meanwhile, the red blood cells after digestion in the examples 1 to 3 are obviously less than those in the comparative examples 1 to 3, and the method can improve the removal rate of the red blood cells and improve the purity of the extracted adipose-derived stem cells; from the number of P0 generation cells, more cells were extracted from the same volume of adipose tissue using the present invention.
Table 4 shows the results of flow identification of examples 1 to 3 and comparative examples 1 to 3
Figure BDA0002623045570000171
And (3) detecting the expression level of the surface marker of the adipose tissue stem cells which are separated and cultured by adopting a flow detection method. As can be seen from the above table, the cells express CD105, CD73, CD90 and CD44 (95% or more), and do not express CD45, CD34, CD14, CD79a, CD19 and HLA-DR (2% or less), which indicates that the identification result of the adipose tissue stem cells cultured by the method of the invention can reach the international identification standard and is better than the standard value. Meanwhile, the invention also provides a loss identification result chart of the embodiment 1, and the graph is shown in an attached figure 5.
Fig. 6 is a photograph showing the effect of direct cleaning and centrifugation after the fat is collected and injected into the bag and transported to the laboratory, and it can be seen from the figure that the bag is obviously four-layer distributed after the cleaning and centrifugation, and the bag is respectively provided with a small amount of grease layer, a fat tissue layer, a cleaning liquid layer and a red blood cell sedimentation layer from top to bottom. As can be seen, the red blood cells in the adipose tissue layer are thoroughly cleaned and removed, and the removal rate of the red blood cells is improved.
FIG. 7 is a graph showing the results of digestion of fat in the digestion bag according to example 1. From the photographs, it can be seen that after digestion for 35min and centrifugation, the inside of the bag is obviously distributed in four layers, namely a fat layer, a small amount of undigested fat tissue layer, a transparent solution layer and a fat stem cell sediment layer from top to bottom. As can be seen from the figure, the bag-type digestion method has excellent effect on the digestion of adipose tissues, and improves the digestion efficiency.
FIG. 8 is a photograph of the layer-by-layer separation and transfer of components using a blood plasma clamp after digestion of adipose tissue. This ensures that the fat remains in a sealed, sterile environment during the transfer process.
The above is only a preferred embodiment of the present invention, and it should be noted that several modifications and improvements made by those skilled in the art without departing from the technical solution should also be considered as falling within the scope of the claims.

Claims (9)

1. The method for extracting the adipose tissue stem cells is characterized by comprising the steps of extracting by adopting a multi-bag adipose tissue stem cell extraction device, wherein the multi-bag adipose tissue stem cell extraction device comprises a first cleaning bag (1), a second cleaning bag (2), a third cleaning bag (3) and a digestion bag (4) which are sequentially connected, and the first cleaning bag (1), the second cleaning bag (2), the third cleaning bag (3) and the digestion bag (4) are communicated with each other through a diversion hose (5); the upper part of the bag body of the digestion bag (4) is provided with two sample inlets and outlets, one sample inlet and outlet is a digestive juice sample inlet and outlet (8), and the other sample inlet and outlet is integrally in a branch shape and comprises a main sample inlet and outlet (9) and two branch sample inlets and outlets (10);
the method comprises the following steps:
(A1) 30mL of adipose tissue active agent (6) is added into the first cleaning bag (1) in advance; the adipose tissue active agent (6) comprises the following components in percentage by mass: 5500.5-1% of hydroxyethyl starch, 02-0.4% of trehalose, 0.8-1.2% of human serum albumin, 0.1% of tribasic sodium citrate and the balance of PBS buffer solution;
(A2) closing a flow guide hose (5) between a first cleaning bag (1) and a second cleaning bag (2), extracting 50-80mL of adipose tissues in an aseptic environment, injecting the adipose tissues into the first cleaning bag (1) through a first sample inlet and outlet (16), and shaking the first cleaning bag (1) to uniformly mix the adipose tissues with the adipose tissue active agent;
(A3) injecting a cleaning solution with the same volume as the adipose tissues from the second sample inlet and outlet (17), and shaking the first cleaning bag (1) to fully and uniformly mix the adipose tissues and the cleaning solution; centrifuging the first cleaning bag (1), wherein the centrifuging speed is 400-600 rpm, the centrifuging time is 5-8min, and the upper layer adipose tissues and a small amount of grease flow into the second cleaning bag (2) through the diversion hose (5);
(A4) injecting a cleaning solution with the same volume as the adipose tissues from the third sample inlet and outlet (18), and shaking the second cleaning bag (2) to fully and uniformly mix the adipose tissues and the cleaning solution; centrifuging the second cleaning bag (2), wherein the centrifuging speed is 400-600 rpm, the centrifuging time is 5-8min, and the upper layer adipose tissues and a small amount of grease flow into the third cleaning bag (3) through the diversion hose (5);
(A5) injecting a cleaning solution with the same volume as the adipose tissues from the fourth sample inlet and outlet (19), and shaking the third cleaning bag (3) to fully and uniformly mix the adipose tissues and the cleaning solution; centrifuging the third cleaning bag (3), wherein the centrifugation speed is 400-600 rpm, and the centrifugation time is 5-8 min; after cleaning and centrifugation, transferring the adipose tissues into the digestion bag (4);
(A6) injecting collagenase I digestive juice with the same volume as the fat tissue in the digestive bag (4) from a digestive juice inlet and outlet (8), then immersing the digestive bag (4) in water bath at 37 ℃, fixing the digestive bag (4) on the bottom plate of a water bath shaker for vibration digestion through magnetic force, carrying out vibration digestion at the rotation speed of 100-200rpm for 30-40min, and observing the digestion condition of the fat tissue at regular time;
(A7) after digestion is finished, injecting a mesenchymal stem cell serum-free complete culture medium with the volume equal to that of liquid in the digestion bag through a branch sample inlet and outlet (10) on the digestion bag (4) to terminate digestion, and after uniform mixing, carrying out centrifugal treatment at the centrifugal rotation speed of 1400-1600rpm for 5-8 min;
(A8) after centrifugation, extracting upper-layer grease, undigested complete adipose tissues and redundant solution from the other branch sample inlet and outlet (10) of the digestion bag (4), and extracting the residual single-cell suspension from the main sample inlet and outlet (9), namely completing the extraction of the adipose tissue stem cells.
2. The method for extracting adipose tissue stem cells according to claim 1, wherein each of the inlet and outlet ports is connected to the inner spaces of the first cleaning bag (1), the second cleaning bag (2), the third cleaning bag (3) and the digestion bag (4) through a guide hose (5).
3. The method for extracting adipose tissue stem cells according to claim 1, wherein the body of the digestion bag (4) is a rounded rectangle, a plurality of strong magnet attachment points (7) are provided on the body, the body is provided with strong magnet blocks through the strong magnet attachment points (7), and the body is fixed on the rocking bed by the magnetic force of the strong magnet blocks and the rocking bed.
4. The method for extracting adipose tissue stem cells according to claim 3, wherein a filter device (11) is provided on the main inlet/outlet port (9).
5. The method for extracting adipose tissue stem cells according to claim 1, wherein the edges of the two sides of the first cleaning bag (1), the second cleaning bag (2), the third cleaning bag (3) and the digestion bag (4) are embedded with a shaped metal strip (12).
6. The method for extracting adipose tissue stem cells according to claim 1, wherein the end of each inlet and outlet sample port is provided with a twist-off sealing rubber ring (13), and the internal interface (14) of each inlet and outlet sample port is adapted to the nipple interface of a syringe.
7. The method for extracting adipose tissue stem cells of claim 1, wherein all the fluid conduits of the inlet and outlet ports are sealed by heat sealing after use.
8. The method for extracting adipose tissue stem cells according to claim 1, wherein the first cleaning bag (1), the second cleaning bag (2), the third cleaning bag (3) and the digestion bag (4) have scales (15) on the surfaces thereof.
9. The method for extracting adipose tissue stem cells according to any of the claims 1 to 8, wherein the material of the diversion hose (5) and the bag body is transparent PVC with high resistance, high toughness, heat-sealability and strong water and oil repellency.
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