CN106554940A - A kind of fat mesenchymal stem cell transport protection liquid - Google Patents
A kind of fat mesenchymal stem cell transport protection liquid Download PDFInfo
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- CN106554940A CN106554940A CN201510614790.7A CN201510614790A CN106554940A CN 106554940 A CN106554940 A CN 106554940A CN 201510614790 A CN201510614790 A CN 201510614790A CN 106554940 A CN106554940 A CN 106554940A
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Abstract
The present invention discloses a kind of fat mesenchymal stem cell transport protection liquid, and the fat mesenchymal stem cell is adjusted in resuspended mode and is added in fat mesenchymal stem cell transport protection liquid, and in transport protection liquid, the concentration of human adipose mesenchymal stem cells is 6 × 105-7×105Individual/ml.It is an advantage of the invention that stability is high, it is easy to storage and transport;Can guarantee that the vigor of cell.
Description
Technical field
The present invention relates to a kind of biological technical field, more particularly to a kind of fat mesenchymal stem cell transport protection liquid suitable for long distance transportation.
Background technology
Inflammatory bowel(IBD)It is higher than Asian countries in Hesperian sickness rate, but the sickness rate of China gradually increases in recent years, digests medical association's survey result of 2007 according to Chinese Medical Association and shows, and Jin10Nian Lai China IBD morbidities number of cases rises 3.1 times, ulcerative colitiss(UC)Prevalence is 11.6/100 ten thousand, clone's engler's disease(CD)About 1.4/100 ten thousand.Crohn disease is a kind of chronic inflammation disease, and pathological changes mainly involve intestinal, and most serious are likely to form fistula, at present the definite cause of disease of not clear this disease, and generally accepted CD pathogenesis are at present:Environment and infective agent act on inheritance susceptible person, in the presence of intestinal microbial population, starts intestinal immune or non-immunological systems, adjusts Intestinal Mucosal Immunity disorderly.Due to the lasting stimulation of antigen, this immunoregulatory disorder shows as excessively hyperfunction and is difficult to, from limit, ultimately result in chronic inflammatory disease and the tissue injury of intestinal mucosa.
After the reported firsts such as 993 years Drakos PE hematopathy merges patient's row stem cell transplantation of CD, can induce CD and alleviate and maintain alleviation for a long time, so as to start the research of stem-cell therapy CD.Mescenchymal stem cell and hematopoietic stem cell have been used to intervene clone's engler's disease at present, and developed country is in development clinical research, and obtains gratifying curative effect, can make fistula and mucosa healing, clone's engler's disease activity index(CDAI)Alleviate, as a result show, for the allogenic mescenchymal stem cell of intractable CD and UC patients(MSCs)Transplantation treatment is safe, effectively, while damaged tissue repair, can also improve the immune system of itself.
But in actual therapeutic, before infusion, cell processing procedure is fairly simple, it cannot be guaranteed that during use cell stability, cytoactive can not be kept long period, therefore it is unsuitable for long distance transportation, mescenchymal stem cell is easily agglomerating in infusion process, after systemic injection fat mesenchymal stem cell, most of stem cell can not reach lesions position so that therapeutic effect is not obvious.And adopt local injection, it is impossible to patients immune system's situation is effectively improved, fundamentally the course of disease is alleviated in face.
The content of the invention
The technical problem to be solved is to provide for a kind of fat mesenchymal stem cell transport protection liquid of suitable long distance transportation, can guarantee that the vigor and stability of cell, and is easy to storage and transport.
In order to solve the technical problem of the above; the invention provides a kind of fat mesenchymal stem cell transport protection liquid; the fat mesenchymal stem cell is adjusted in resuspended mode and is added in fat mesenchymal stem cell transport protection liquid, and in transport protection liquid, the concentration of human adipose mesenchymal stem cells is 6 × 105-7×105Individual/ml.
Fat mesenchymal stem cell transport preserves liquid and is made up of the glucose for injection liquid of Human Albumin stock solution and 9.5%-40% of 30%-50%, the injection normal saline of 9.5%-40%, 0.5% heparin sodium.
The concentration of the Human Albumin stock solution is 10%.
The fat mesenchymal stem cell is adjusted in resuspended mode and is added in fat mesenchymal stem cell transport protection liquid, is comprised the following steps:
1. a little cell suspension is drawn with micro sample-adding rifle, be used as cell counting and cell viability detection, give up dead cell, and living cell counting;If viable count concentration is 6 × 105-7×1053. individual cell/1ml, carry out step;Otherwise, 2. continuation step is adjusted;
2. it is centrifuged 5 minutes with the centrifugal speed of 800rpm, supernatant is abandoned in suction, adds 2-6 milliliters transport protection liquid, make cell concentration be 6 × 105-7×105Individual cell/1ml, then carry out step 3.;
3. by adjustment after 6 × 105-7×105The human adipose mesenchymal stem cells transport protection liquid of individual cell/1ml is packaged preservation, as shown in Figure 2.
The cell counting and cell viability detection, are that the platform of cell suspension and 0.4% being expected, blue solution, by 1: 1 mixing, after mixing 1 minute, counts cell with blood counting chamber under the microscope;Living cells repel platform and expect orchid, and the cell for dying blueness is dead cell.
The human adipose mesenchymal stem cells, its preparation method include the steps:
(1) pick and place culture bottles of the 3-5 for human adipose mesenchymal stem cells;
(2) culture bottle cap mouth surrounding, bottle cap of then outwarding winding are wiped with cotton ball soaked in alcohol;
(3) inhale and abandon original culture medium in culture bottle, per bottle is cleaned with the phosphate buffer of 10ml twice, and washing liquid is abandoned in suction;(4) 1ml pancreas enzyme -EDTAs are added in per bottle, is put into 2-3 minutes in 37 DEG C of incubators;(5) observation of cell under inverted microscope, gently patting culture bottle with palm guarantees that human adipose mesenchymal stem cells catapepsises are got off;(6) culture medium of the 1ml containing serum is added in per bottle to terminate digestion;The phosphate buffer of 10ml is added in per bottle, is blown and beaten repeatedly, cell suspension is obtained;(7) cell suspension is moved in centrifuge tube, is centrifuged 5 minutes with the centrifugal speed of 800rpm, centrifugation terminates, supernatant is abandoned in suction, obtain human adipose mesenchymal stem cells, as shown in Figure 1.
The fat mesenchymal stem cell transport protection liquid for preparing needs the storage and transport under 4-8 DEG C of light protected environment, and effect duration is 12 hours from the subpackage.
The present invention superior effect be:
1)The fat mesenchymal stem cell transport protection liquid stability that the application is prepared is high, is easy to preserve and long distance transportation, using safer, can be clinically to use;
2)The vigor and stability of cell is can guarantee that, effectively can be played a role in vivo, prevent from cloning the recurrent exerbation of engler's disease.
Description of the drawings
The Figure of description for constituting the part of the application is used for providing a further understanding of the present invention, and the schematic description and description of the present invention is used to explain the present invention, does not constitute inappropriate limitation of the present invention.In the accompanying drawings:
Human adipose mesenchymal stem cells figures under the microscope of the Fig. 1 for the application;
Fat mesenchymal stem cell transport protection liquid figures under the microscope of the Fig. 2 for the application;
Fig. 3 is CDAI index variation figures after the application specific embodiment patient treatment.
Specific embodiment
Embodiments of the invention are described in detail below in conjunction with accompanying drawing, but the multitude of different ways enforcement that the present invention can be defined by the claims and cover.
Describe embodiments of the invention below in conjunction with the accompanying drawings in detail.
This application provides a kind of fat mesenchymal stem cell transport protects liquid, the fat mesenchymal stem cell to adjust in resuspended mode and be added in fat mesenchymal stem cell transport protection liquid, in transport protection liquid, the concentration of human adipose mesenchymal stem cells is 6 × 105-7×105Individual/ml.Stability is high, is easy to preserve and long distance transportation, using safer, can be clinically to use.
Fat mesenchymal stem cell transport protection liquid is made up of the glucose for injection liquid of Human Albumin stock solution and 9.5%-40% of 30%-50%, the injection normal saline of 9.5%-40%, 0.5% heparin sodium.
Specific embodiment 1:Fat mesenchymal stem cell transport protection liquid by 50% Human Albumin stock solution, 9.5% glucose for injection liquid, 40% injection normal saline and 0.5% heparin sodium constitute.
Specific embodiment 2:Fat mesenchymal stem cell transport protection liquid by 50% Human Albumin stock solution, 29.5% glucose for injection liquid, 20% injection normal saline and 0.5% heparin sodium constitute.
Specific embodiment 3:Fat mesenchymal stem cell transport protection liquid by 30% Human Albumin stock solution, 30% glucose for injection liquid, 39.5% injection normal saline and 0.5% heparin sodium constitute.
The Human Albumin stock solution is the Human Albumin stock solution of the Chinese medicines quasi-word for S10920005 of Haerbin Shiheng Bioengineering Medicine Stock Co., Ltd.'s production, and the original liquid concentration is 10%.
The fat mesenchymal stem cell is adjusted in resuspended mode and is added in fat mesenchymal stem cell transport protection liquid, is comprised the following steps:
1. a little cell suspension is drawn with micro sample-adding rifle, be used as cell counting and cell viability detection, give up dead cell, and living cell counting;If viable count concentration is 6 × 105-7×1053. individual cell/1ml, carry out step;Otherwise, 2. continuation step is adjusted;
2. it is centrifuged 5 minutes with the centrifugal speed of 800rpm, supernatant is abandoned in suction, adds 2-6 milliliters transport protection liquid, make cell concentration be 6 × 105-7×105Individual cell/1ml, then carry out step 3.;
3. by adjustment after 6 × 105-7×105The human adipose mesenchymal stem cells transport protection liquid of individual cell/1ml is packaged preservation, as shown in Figure 2.
The cell counting and cell viability detection, are that the platform of cell suspension and 0.4% being expected, blue solution, by 1: 1 mixing, after mixing 1 minute, counts cell with blood counting chamber under the microscope;Living cells repel platform and expect orchid, and the cell for dying blueness is dead cell.
The human adipose mesenchymal stem cells, its preparation method include the steps:
(1) pick and place culture bottles of the 3-5 for human adipose mesenchymal stem cells;
(2) culture bottle cap mouth surrounding, bottle cap of then outwarding winding are wiped with cotton ball soaked in alcohol;
(3) inhale and abandon original culture medium in culture bottle, per bottle is cleaned with the phosphate buffer of 10ml twice, and washing liquid is abandoned in suction;(4) 1ml pancreas enzyme -EDTAs are added in per bottle, is put into 2-3 minutes in 37 DEG C of incubators;(5) observation of cell under inverted microscope, gently patting culture bottle with palm guarantees that human adipose mesenchymal stem cells catapepsises are got off;(6) culture medium of the 1ml containing serum is added in per bottle to terminate digestion;The phosphate buffer of 10ml is added in per bottle, is blown and beaten repeatedly, cell suspension is obtained;(7) cell suspension is moved in centrifuge tube, is centrifuged 5 minutes with the centrifugal speed of 800rpm, centrifugation terminates, supernatant is abandoned in suction, obtain human adipose mesenchymal stem cells, as shown in Figure 1.
The fat mesenchymal stem cell transport protection liquid for preparing needs the storage and transport under 4-8 DEG C of light protected environment, and effect duration is 12 hours from the subpackage.
The fat mesenchymal stem cell transport protection liquid that the application is prepared is used for the patient with late period clone engler disease; when seeing and treating patients, CDAI coefficients are 305; long-time abdominal pain diarrhea; whole body electrolyte disturbance; after seeing and treating patients; the a small amount of fatty tissue of patient is obtained, amplification cultivation is separated in vitro and is obtained substantial amounts of fat mesenchymal stem cell, adult adipose-derived mesenchymal stem injection is prepared by the application.Patient is subsequently according to the requirement of intravenous injection; systemic injection fat mesenchymal stem cell transport protection liquid; three times is a course for the treatment of; every minor tick one month; simultaneously when fistula revisional operation is carried out; the fat mesenchymal stem cell transport protection liquid of local injection itself, patient follow-up CADI within treatment 6 months is received score all<150, as shown in figure 3, entering the catabasises, the healing of patient's fistula is good, and without recurring, the mental status is good, and abdominal pain diarrhea disappears.
In sum, the fat mesenchymal stem cell transport protection liquid energy that the application is prepared ensures the vigor and stability of cell, can effectively alleviate the state of an illness, prevent the recurrent exerbation of symptom.
The preferred embodiments of the present invention are the foregoing is only, the present invention is not limited to, for a person skilled in the art, the present invention there can be various modifications and variations.All any modification, equivalent substitution and improvements within the spirit and principles in the present invention, made etc., should be included within the scope of the present invention.
Claims (6)
1. liquid is protected in a kind of fat mesenchymal stem cell transport, it is characterised in that:The fat mesenchymal stem cell is adjusted in resuspended mode and is added in fat mesenchymal stem cell transport protection liquid, and in transport protection liquid, the concentration of human adipose mesenchymal stem cells is 6 × 105-7×105Individual/ml.
2. liquid is protected in the fat mesenchymal stem cell transport according to claims 1, it is characterised in that:Fat mesenchymal stem cell transport preserves liquid and is made up of the glucose for injection liquid of Human Albumin stock solution and 9.5%-40% of 30%-50%, the injection normal saline of 9.5%-40%, 0.5% heparin sodium.
3. liquid is protected in the fat mesenchymal stem cell transport according to claims 2, it is characterised in that:The concentration of the Human Albumin stock solution is 10%.
4. liquid is protected in the fat mesenchymal stem cell transport according to claims 1, it is characterised in that:The fat mesenchymal stem cell is adjusted in resuspended mode and is added in fat mesenchymal stem cell transport protection liquid, is comprised the following steps:
1. a little cell suspension is drawn with micro sample-adding rifle, be used as cell counting and cell viability detection, give up dead cell, and living cell counting;If viable count concentration is 6 × 105-7×1053. individual cell/1ml, carry out step;Otherwise, 2. continuation step is adjusted;
2. it is centrifuged 5 minutes with the centrifugal speed of 800rpm, supernatant is abandoned in suction, adds transport protection liquid, makes cell concentration be 6 × 105-7×105Individual cell/1ml, then carry out step 3.;
3. by adjustment after 6 × 105-7×105The human adipose mesenchymal stem cells transport protection liquid of individual cell/1ml is packaged preservation.
5. liquid is protected in the fat mesenchymal stem cell transport according to claims 4, it is characterised in that:The cell counting and cell viability detection, are that the platform of cell suspension and 0.4% being expected, blue solution, by 1: 1 mixing, after mixing 1 minute, counts cell with blood counting chamber under the microscope;Living cells repel platform and expect orchid, and the cell for dying blueness is dead cell.
6. liquid is protected in the fat mesenchymal stem cell transport according to claims 1, it is characterised in that:The human adipose mesenchymal stem cells, its preparation method include the steps:
(1) pick and place culture bottles of the 3-5 for human adipose mesenchymal stem cells;
(2) culture bottle cap mouth surrounding, bottle cap of then outwarding winding are wiped with cotton ball soaked in alcohol;
(3) inhale and abandon original culture medium in culture bottle, per bottle is cleaned with the phosphate buffer of 10ml twice, and washing liquid is abandoned in suction;(4) 1ml pancreas enzyme -EDTAs are added in per bottle, is put into 2-3 minutes in 37 DEG C of incubators;(5) observation of cell under inverted microscope, gently patting culture bottle with palm guarantees that human adipose mesenchymal stem cells catapepsises are got off;(6) culture medium of the 1ml containing serum is added in per bottle to terminate digestion;The phosphate buffer of 10ml is added in per bottle, is blown and beaten repeatedly, cell suspension is obtained;(7) cell suspension is moved in centrifuge tube, is centrifuged 5 minutes with the centrifugal speed of 800rpm, centrifugation terminates, supernatant is abandoned in suction, obtain human adipose mesenchymal stem cells.
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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CN107912420A (en) * | 2017-10-27 | 2018-04-17 | 北京协科医药科技有限公司 | A kind of cell preservation method, preserve liquid and preserve liquid and preparation method thereof |
CN107912424A (en) * | 2017-12-01 | 2018-04-17 | 佛山科学技术学院 | A kind of special room-temperature extender of dog mescenchymal stem cell and application |
CN108056095A (en) * | 2017-12-28 | 2018-05-22 | 重庆斯德姆生物技术有限公司 | A kind of fat mesenchymal stem cell transport protection liquid and its application |
CN108990966A (en) * | 2018-08-24 | 2018-12-14 | 上海中溢精准医疗科技有限公司 | A kind of umbilical cord mesenchymal stem cells transport protection liquid and its guard method |
CN109845725A (en) * | 2019-01-25 | 2019-06-07 | 北京益华生物科技有限公司 | Cell transport saves liquid and its application |
CN110199987A (en) * | 2019-07-22 | 2019-09-06 | 深圳市未来细胞生命科技有限公司 | A kind of mesenchyme stem cell preserving fluid and its store method and application |
CN111418579A (en) * | 2020-04-13 | 2020-07-17 | 广东华夏健康生命科学有限公司 | Preservation method of adipose tissues, preservation solution of adipose tissues and preparation method of preservation solution |
CN112075418A (en) * | 2020-09-29 | 2020-12-15 | 广州同康生物科技有限公司 | Adipose mesenchymal stem cell cryopreservation liquid and adipose mesenchymal stem cell cryopreservation method |
CN113498779A (en) * | 2021-04-22 | 2021-10-15 | 重庆医科大学附属儿童医院 | Reagent and method for transporting cells |
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Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
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CN107912420A (en) * | 2017-10-27 | 2018-04-17 | 北京协科医药科技有限公司 | A kind of cell preservation method, preserve liquid and preserve liquid and preparation method thereof |
CN107912424A (en) * | 2017-12-01 | 2018-04-17 | 佛山科学技术学院 | A kind of special room-temperature extender of dog mescenchymal stem cell and application |
CN108056095A (en) * | 2017-12-28 | 2018-05-22 | 重庆斯德姆生物技术有限公司 | A kind of fat mesenchymal stem cell transport protection liquid and its application |
CN108990966A (en) * | 2018-08-24 | 2018-12-14 | 上海中溢精准医疗科技有限公司 | A kind of umbilical cord mesenchymal stem cells transport protection liquid and its guard method |
CN109845725A (en) * | 2019-01-25 | 2019-06-07 | 北京益华生物科技有限公司 | Cell transport saves liquid and its application |
CN110199987A (en) * | 2019-07-22 | 2019-09-06 | 深圳市未来细胞生命科技有限公司 | A kind of mesenchyme stem cell preserving fluid and its store method and application |
CN111418579A (en) * | 2020-04-13 | 2020-07-17 | 广东华夏健康生命科学有限公司 | Preservation method of adipose tissues, preservation solution of adipose tissues and preparation method of preservation solution |
CN111418579B (en) * | 2020-04-13 | 2021-05-18 | 广东华夏健康生命科学有限公司 | Preservation method of adipose tissues, preservation solution of adipose tissues and preparation method of preservation solution |
CN112075418A (en) * | 2020-09-29 | 2020-12-15 | 广州同康生物科技有限公司 | Adipose mesenchymal stem cell cryopreservation liquid and adipose mesenchymal stem cell cryopreservation method |
CN112075418B (en) * | 2020-09-29 | 2021-10-22 | 惟力维斯(上海)医学科技有限公司 | Adipose mesenchymal stem cell cryopreservation liquid and adipose mesenchymal stem cell cryopreservation method |
CN113498779A (en) * | 2021-04-22 | 2021-10-15 | 重庆医科大学附属儿童医院 | Reagent and method for transporting cells |
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