CN102920734A - Mesenchymal stem cell injection and preparation method thereof as well as application in preparation of medicine for treating ulcerative colitis - Google Patents
Mesenchymal stem cell injection and preparation method thereof as well as application in preparation of medicine for treating ulcerative colitis Download PDFInfo
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Abstract
The invention discloses a mesenchymal stem cell injection and a preparation method of the mesenchymal stem cell injection and an application in preparation of a medicine for treating ulcerative colitis, wherein the mesenchymal stem cell injection consists of the following components: 2*105-1*107 pieces/ml of mesenchymal stem cells, 5-10% of dimethyl sulfoxide (DMSO) by volume, 1-6% of human albumin by mass-to-volume ratio, and 1% of low molecular dextran and compound electrolyte solution. According to the mesenchymal stem cell injection, the preparation method and the application of the mesenchymal stem cell injection in preparation of the medicine for treating ulcerative colitis, the mesenchymal stem cell injection is used for correcting immune dysfunction of a patient of ulcerative colitis, and preventing the autoimmune dysfunction from continuously damaging the intestinal tract; meanwhile, many cell growth factors and repair factors are secreted, to promote pathologically changed intestinal ulcer to heal, therefore, the purpose of foundational treating the ulcerative colitis is achieved. The disease course of the ulcerative colitis can be reversed, the patient can be helped in escaping out of influence of stomachache and diarrhea on the life as well as toxic and side effects of the medicine, so as to treat the ulcerative colitis thoroughly.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of mescenchymal stem cell injection and preparation method thereof and the application in preparation treatment ulcerative colitis medicine.
Background technology
Ulcerative colitis can betide any age level
But be more common in 20-40 year.Developed country and city are more than the rural area
Pathogenic factor it be unclear that, and thinks at present immunologic derangement, and the combined effect of nature-nurture factor causes morbidity.Research finds that ulcerative colitis easily betides some specific family
The one-level relative of about 20% left and right sides patients of ulcerative colitis (i.e. hall/cousins/sister or more get close to) also suffer from ulcerative colitis, so inherited genetic factors has played certain effect.Except the gastrointestinal symptom
The symptom at other positions of body can appear in some patient, comprising: eyes are red and itch swollen; Oral ulcer; Arthralgia; Phyma and other damages; Osteoporosis; Renal calculus etc.There have the patient of ulcerative colitis 8-10 medical history to suffer from the colon cancer risk to be higher.
Ulcerative colitis is different from general inflammation disease, patient's intestinal mucosa damage is serious, only having could rehabilitation to its reparation patient who carries out fundamentally, what the means such as operation traditionally, medicine were alleviated is surperficial inflammatory reaction, cure the symptoms, not the disease, so therapeutic effect is undesirable, the state of an illness still can recur, and can't allow patient and family members satisfied.
Mescenchymal stem cell (MSCs) is the important member of stem cell family, because it has the concern that multi-lineage potential, hematopoiesis support and the characteristics such as the implantation of promotion stem cell and self replication are subject to people day by day.MSCs has reduced immunogenicity and unique immunoregulation effect, can control agent in too drastic or weak immunoreation, reduce self abnormal immune reaction to the destruction of autologous tissue.And MSCs can be divided into the Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium in vivo or under the external specific inductive condition, continuous passage cultivate and freezing preservation after still have multi-lineage potential.The MSCs in Placenta Hominis, umbilical cord source has that differentiation potential is large, multiplication capacity is strong, immunogenicity is low, draws materials conveniently, the restriction of amoral ethical issues, be easy to the feature such as preparation of industrialization, makes it might become pluripotent stem cell for the tool potential applicability in clinical practice of the adjusting of tissue repair, immunologic derangement and metabolic disease cell therapy.
What the treatment of ulcerative colitis mainly solved at present is to eliminate symptom and keep asymptomatic state, can not fundamentally treat disease, the patient needs strictly to keep on a diet throughout one's life, in the case still easily recurrence, stomachache, diarrhoea and mucopurulent bloody stool have a strong impact on Quality of Life, and the drug side effectes such as hormone and the pyridine of willow nitrogen sulphur arsenic are very large.Along with the progress of the course of disease, the complication of ulcerative colitis engenders and increases the weight of that the patient that medical history is long is prone to colon cancer.Present medicine and operative treatment can not well be avoided the generation of these situations, adjust the immunologic derangement in the patients of ulcerative colitis body, and the intestinal segment of repairing pathological changes is only the basic for the treatment of ulcerative colitis.
The long refractory of the ulcerative colitis course of disease more, can impel the colonic pathological change healing by medicine, alleviate diarrhoea, rectum is had blood in stool and the symptom such as stomachache, reach and eliminate symptom and keep asymptomatic state, but can not treat disease from the cause of disease, and some patients were is because the pathological changes intestinal segment is extensive, Drug therapy can not mitigate the disease, must treat by excision pathological changes intestinal segment, surgical effect still can, but the complication of operation is more, comprises severe haemorrhage that ulcer causes, intestinal perforation, toxic megacolon, canceration of ulcer etc., and risk is larger.In view of present pathogenesis of ulcerative colitis rate increases and year by year without the easy Therapeutic Method of specially good effect, the present invention intends fundamentally solving the predicament of patient's Long-term taking medicine and Relapse rate outbreak, treatment ulcerative colitis.
The shortcoming of prior art: the at present treatment of ulcerative colitis mainly depends on the Drug therapys such as diet control, the arsenic pyridine of willow nitrogen sulphur and hormone, herbal enema, needs operative treatment when being in a bad way.The symptoms such as stomachache, diarrhoea and bloody purulent stool can be temporarily controlled in the integrated use of these therapies, can not effectively correct immunologic derangement in the patient body, can not fundamentally treat progress and the recurrence of ulcerative colitis and the prevention state of an illness.And the medicines such as sulfasalazine and hormone have very serious toxic and side effects, such as: anaphylaxis, neutrophilic granulocyte minimizing or deficiency disease, thrombocytopenia and aplastic anemia, hemolytic anemia and hemoglobinuria, liver injury etc.This disease can be partly cured in operation, but post-operative complication is more and more serious, as: adhesive ileus, Anastomotic bleeding, fistula etc. have a strong impact on Quality of Life.
Summary of the invention
For the defects that ulcerative colitis in the prior art exists, the invention provides a kind of mescenchymal stem cell injection and preparation method thereof and the application in preparation treatment ulcerative colitis medicine.Described injection can fundamentally be regulated the immunologic derangement of patients of ulcerative colitis, stops autoimmune disorder to the destruction of intestinal tissue; Promote the reparation and the healing of ulcer of pathological changes intestinal, thereby toxicity and the state of an illness that the medicines such as puzzlement, hormone that make the patient break away from abdominal pain diarrhea bring are controlled the not good severe complication that causes.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
A kind of mescenchymal stem cell injection, it comprises following component:
Content is 2 * 10
5-1 * 10
7The mescenchymal stem cell of/ml;
Volume ratio is the clinical grade DMSO of 5-10%;
Mass volume ratio is the human albumin of 1-6%;
Mass volume ratio is 1% low molecular dextran;
Surplus is compound electrolyte solution.
To further improvement in the technical proposal: described source for mesenchymal stem cells is in people's umbilical cord and/or Placenta Hominis.
To further improvement in the technical proposal: described mescenchymal stem cell vigor remains on more than 85%.
The present invention also provides the preparation method of described mescenchymal stem cell injection, comprises the preparation of umbilical cord mesenchymal stem cells and the preparation of placenta mesenchyma stem cell.
The present invention also provides the application of described mescenchymal stem cell injection in preparation treatment ulcerative colitis medicine.
To further improvement in the technical proposal: the consumption of described mescenchymal stem cell injection is for containing 1 * 10
6-1 * 10
8Individual/the ml mescenchymal stem cell.
Compared with prior art, advantage of the present invention and good effect are:
1. the present invention selects to prepare the mescenchymal stem cell injection with the mescenchymal stem cell that derives from people's umbilical cord and Placenta Hominis, and described mescenchymal stem cell output is larger, and the preparation system is easy to Quality Control, is easy to industrialization.
2. the mescenchymal stem cell injectable liquefied composition is comprised of human mesenchymal stem cell, clinical grade DMSO, human albumin, low molecular dextran and vigorous arteries and veins power (compound electrolyte solution).This injection can be directly frozen through program control cooling, can be directly used in clinical injection after the recovery.
3. the present invention utilizes the human mesenchymal stem cell injection to correct the interior immunologic derangement of patients of ulcerative colitis body, treats disease from the cause of disease, stops autoimmune disorder that the continuation of intestinal is destroyed; Secrete simultaneously a lot of cell growth factor and reparative factor, promote the healing of pathological changes intestinal segment ulcer, thereby reach the purpose of fundamentally treating ulcerative colitis.The present invention can reverse the ulcerative colitis course of disease, stops the appearance of complication, helps the patient to break away from abdominal pain diarrhea to the toxic and side effects that impact and the medicine of life brings, and thoroughly treats ulcerative colitis.
After reading the specific embodiment of the present invention by reference to the accompanying drawings, other characteristics of the present invention and advantage will become clearer.
Description of drawings
Fig. 1 is the microphotograph of human umbilical cord mesenchymal stem cells described in the present invention.
Fig. 2 changes comparison diagram behind the rat colon tissue treatment among the present invention.
Fig. 3 is pathological change comparison diagram behind the rat colon tissue treatment among the present invention.
Fig. 4 is that the rear rat blood serum TNF-alpha content for the treatment of changes comparison diagram among the present invention.
The specific embodiment
Below in conjunction with the drawings and specific embodiments technical scheme of the present invention is described in further detail.
Embodiment 1
One, the preparation of umbilical cord mesenchymal stem cells
1. fresh mature healthy fetal cord washes with the PBS buffer, and described PBS buffer contains 100 kU/ L penicillins and 100 mg/ L streptomycins;
2. the long umbilical cord of clip 5-10cm is cut into the long segment of 2cm with umbilical cord, repeatedly washes with the PBS buffer; Described PBS buffer contains 100 kU/ L penicillins and 100 mg/ L streptomycins;
3. the umbilical cord tissue piece is shredded into 2-5mm
3Fritter; Add the washing of L-DMEM culture medium, under the 500-800g condition centrifugal 5 minutes, abandon supernatant;
4. with piece of tissue and by volume 2.5-3:1 ratio adding of culture medium culture medium, the mixing piece of tissue is seeded in the Tissue Culture Dish, puts incubator and cultivates; Described culture medium is for containing the special-purpose serum-free medium of 1-10ng/ml basic fibroblast growth factor (bFGF) and MSC;
5. changed a subculture in per 3 days, went down to posterity when cell reached about 80% fusions to 8-9 days; The culture medium that goes down to posterity is the special-purpose serum-free medium of MSC.
Two, the preparation of placenta mesenchyma stem cell
1. with the PBS buffer flushing Placenta Hominis that contains 100 kU/ L penicillins, 100 mg/ L streptomycins and 50u/ml heparin sodium, along placental edge fetus face amniotic membrane is slowly peeled off;
2. again wash Placenta Hominis source amniotic membrane, amniotic membrane is cut into 1-5mm
3Fritter;
3. with the amnion tissue piece DMEM culture medium mixing that contains penicillin and streptomycin, centrifugal 10min under the 700-950g condition;
4. abandon supernatant, every pipe adds and contains volume ratio 0.25% tryptic DMEM cultivation based on 37 ℃ of digestion 10min, centrifugal 10min under the 700-950g condition;
5. abandon supernatant, after every pipe adds complete culture medium, centrifugal 10min under the 2200rpm condition; Described complete medium is the MSC serum-free medium that contains 100 kU/ L penicillins+100 mg/ L streptomycins;
6. abandon supernatant, the amnion tissue piece is inoculated in the culture dish, adds complete culture medium, cultivates at incubator; Carry out half amount in per 3 days and change liquid;
7. the cell attachment growth was arranged to 10-12 days, behind the cell clonal formation, discard piece of tissue, add complete culture medium and cultivate;
8. merge to 80-90% to 15-17 days cell clonies, carry out passage.
Three, the preparation of mescenchymal stem cell injection
This injection is formulated by following components in certain proportion:
1, derive from people's umbilical cord and/or placenta mesenchyma stem cell, the quantity of mescenchymal stem cell is 2 * 10 in every milliliter of injection
5-1 * 10
7Individual;
2, volume ratio is the DMSO(clinical grade of 5-10%);
3, mass volume ratio is the human albumin of 1-6%;
4, mass volume ratio is 1% low molecular dextran
5, surplus is vigorous arteries and veins power (compound electrolyte solution).
Clinical grade DMSO is nontoxic to cell, can play better the effect of Cell protection, need not rinsing behind cell recovery, can be directly used in clinical injection, and is nontoxic to patient safety.
The human albumin is clinical injection commonly used, can be cell nutrition is provided, and is beneficial to the metabolism of cell.
The interpolation assurance cell of 1% low molecular dextran is kept good cell dispersity in the preservation process, reduced the intercellular phenomenon agglomerating and the cell adhesion chamber wall of sticking, the danger of the agglomerating thromboembolism of contingent blood vessel inner cell when having reduced clinical cell infusion has simultaneously also reduced cell aggregation and has been filtered the loss cell that causes by filter for infusion.
Vigorous arteries and veins power (compound electrolyte solution) can keep the osmotic pressure of cell, is beneficial to the survival of cell.This injection component is clinical common electrolyte solution, can make things convenient for clinical infusion.
This injection can be stablized preservation in-196 ℃ of temperature.But direct infusion is in patient body after the recovery of thawing during use, and cell remains the single cell suspension state, and cell viability remains on more than 85%, and can not cause the uncomfortable reaction of patient.
This injection is beneficial to preservation, transportation and the survival of mescenchymal stem cell very much, and clinical infusion safety, cell can keep higher vigor for a long time in this injection, be convenient to transport and not limited by the harshness of service time, solved the strange land patient when using cell transport for a long time the problem that affects cell viability.
The concrete process for preparation of mescenchymal stem cell injection is: such as preparation 100ml stem cell injection, this injection is by human mesenchymal stem cell, clinical grade DMSO 5ml, human albumin's stock solution (the stock solution quality volume by volume concentration is 20%) 10ml, low molecular dextran 1ml(original liquid concentration 100%) and vigorous arteries and veins power (compound electrolyte solution) 84ml form.The ratio of the equal representation quality of mass volume ratio of the present invention (g) and volume (ml).The quantity of mescenchymal stem cell is 2 * 10 in every milliliter of injection
5-1 * 10
7The mescenchymal stem cell injection still can keep the single cell suspension state in-196 ℃ of ambient temperatures, cell viability remains on more than 85% after the recovery, can be directly used in clinical injection.
Four, the mescenchymal stem cell injection is to safety and the effectiveness experiment of ulcerative colitis in rats treatment
45 of healthy cleaning level SD rats, are divided into 3 groups, 15 every group at random at 6-8 age in week.Adopt immune composite algorithm to prepare Ulcerative Colitis Model.Two groups of rats of A, B are all in testing 1 day, 14 days at its groin, toes place injection emulsion 0.4ml.Fasting (can't help water) 24h after the immunity, 3% chloral hydrate intraperitoneal injection of anesthesia, per anum push 0.6ml liquid (100mg/kgTNBS+50% ethanol).A group rat (contains 5 * 10 through tail vein injection 1ml human mesenchymal stem cell injection behind the modeling 24h
6Cell, electromicroscopic photograph are as shown in Figure 1), B group rat is through tail vein injection 1ml PBS.The C group is normal SD rats.
Experiment begins regularly to observe activities in rats and sign rear every day.All treat anesthesia in rear 3 days, 7 days, 14 days in mescenchymal stem cell for 3 groups and put to death 5 rats, cut open and get colon, observe Traumatic Colon, and examine under a microscope the colon pathological change.
The result: whole experimentation has no rat and acute shock or death occur.
1. the rat animation is observed: after the modeling 1 day, A, B group rat namely begin to occur anorexia, lazy moving, times of defecation increases, soft stool or loose stool etc.Symptom reaches the peak after 3 days, and symptom begins to alleviate afterwards, and A organizes alleviation speed and organizes faster than B, and the A group was without diarrhoea when experiment finished, and activity is normal.The C group has no any abnormal symptom.
2. colon changes: treated rear 3 days, A, B group sees all that the colon intestinal wall obviously thickens, congestion and edema, ulcer that the subregion visible range is larger; Treated rear 7 days, A group congestion and edema slightly alleviates, and the ulcer scope also reduces, and B group pathological changes is without remarkable improvement; Treated rear 14 days, the rarely seen mild hyperaemia edema of A group colon has no ulcer, B group pathological changes slightly alleviates, experimental result as shown in Figure 2, left figure is: A group Rat Mesenchymal Stem Cells is treated rear 3 days colons, the colon intestinal wall obviously thickens, congestion and edema, ulcer that the subregion visible range is larger; Middle figure is: A group Rat Mesenchymal Stem Cells is treated rear 14 days colons, and pathological changes obviously alleviates, ulcer healing; Right figure is: B group rat is treated rear 14 days colons, still has obvious congestion and edema and macroscopic ulcer.
3. colon's pathological change: behind the cell therapy 3 days, A group colon pathological section HE dyeing microscopically was seen epithelial defect in a big way, and mucosa and tela submucosa have inflammatory cell infiltration, and B group pathological change is similar to the A group; Treated rear 7 days, A group epithelial defect scope reduces to some extent, and mucosa and tela submucosa are seen a large amount of inflammatory cell infiltrations, and B group pathological changes is without remarkable improvement; To 14 days, A group mucosa structure was substantially complete, and a small amount of inflammatory cell infiltration is only arranged, experimental result as shown in Figure 3, left figure is: A group Rat Mesenchymal Stem Cells is treated rear 3 days colon's pathology, visible epithelial defect in a big way under the mirror, and mucosa and tela submucosa have inflammatory cell infiltration; Middle figure is: A group Rat Mesenchymal Stem Cells is treated rear 14 days colon's pathology, and visible mucosa structure is substantially complete under the mirror, and a small amount of inflammatory cell infiltration is only arranged; Right figure is: B group rat is treated rear 14 days colon's pathology, still has large-scale epithelial defect under the mirror, and mucosa and tela submucosa have inflammatory cell infiltration.
4. immune indexes changes in the rat body: behind the cell therapy 3 days, A group rat blood serum TNF-alpha content obviously raise than matched group, and the B group changes and is similar to the A group; Treated rear 7 days, A group rat blood serum TNF-alpha content reduces to some extent, and B group pathological changes is without remarkable improvement; To 14 days, A group rat blood serum TNF-alpha content obviously reduced than the B group, and experimental result was treated rear 14 days as shown in Figure 4, and A group rat blood serum TNF-alpha content obviously reduces (P＜0.05) than the B group.
Conclusion: human mesenchymal stem cell injection for treating ulcerative colitis in rats safe and feasible, animation, symptom and sign, colon pathology and the immunologic derangement state of ill rat all take an evident turn for the better after the mescenchymal stem cell treatment, the treatment significant effective.
Embodiment 2
As prepare 100ml stem cell injection, this injection is comprised of human mesenchymal stem cell, clinical grade DMSO 10ml, human albumin's stock solution (original liquid concentration is 20%) 10ml, low molecular dextran 1ml and vigorous arteries and veins power (compound electrolyte solution) 79ml.The quantity of mescenchymal stem cell is 2 * 10 in every milliliter of injection
5-1 * 10
7The mescenchymal stem cell injection still can keep the single cell suspension state in-196 ℃ of ambient temperatures, cell viability remains on more than 85% after the recovery, can be directly used in clinical injection.
Above embodiment is only in order to illustrating technical scheme of the present invention, but not limits it; Although with reference to previous embodiment the present invention is had been described in detail, for the person of ordinary skill of the art, still can make amendment to the technical scheme that previous embodiment is put down in writing, perhaps part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution break away from the spirit and scope of the present invention's technical scheme required for protection.
Claims (8)
1. mescenchymal stem cell injection is characterized in that it comprises following component:
Content is 2 * 10
5-1 * 10
7The mescenchymal stem cell of individual/ml;
Volume ratio is the clinical grade DMSO of 5-10%;
Mass volume ratio is the human albumin of 1-6%;
Mass volume ratio is 1% low molecular dextran;
Surplus is compound electrolyte solution.
2. mescenchymal stem cell injection according to claim 1, it is characterized in that: described source for mesenchymal stem cells is in people's umbilical cord and/or Placenta Hominis.
3. mescenchymal stem cell injection according to claim 1, it is characterized in that: described mescenchymal stem cell vigor remains on more than 85%.
4. the preparation method of mescenchymal stem cell injection according to claim 1, it is characterized in that dose volume is 1% low molecular dextran and compound electrolyte solution than the human albumin, the mass volume ratio that are 1-6% for the DMSO of 5-10%, mass volume ratio, mescenchymal stem cell is resuspended in makes single cell suspension in the mentioned solution, making stem cell population is 2 * 10
5-1 * 10
7Individual/ml.
5. the preparation method of mescenchymal stem cell injection according to claim 4 is characterized in that the preparation of described umbilical cord mesenchymal stem cells may further comprise the steps:
(1). get fresh mature healthy fetal cord, with the PBS buffer flushing that contains 100kU/L penicillin and 100mg/L streptomycin;
(2). the long umbilical cord of clip 5-10cm, umbilical cord is cut into the long segment of 2cm, repeatedly wash with described PBS buffer;
(3). the umbilical cord tissue piece is shredded into 2-5mm
3Fritter; Add the washing of L-DMEM culture medium, under the 500-800g condition centrifugal 5 minutes, abandon supernatant;
(4). with piece of tissue and by volume 2.5-3:1 ratio adding of culture medium culture medium, the mixing piece of tissue is seeded in the Tissue Culture Dish and cultivates, and described culture medium is the special-purpose serum-free medium of MSC that contains the 1-10ng/ml basic fibroblast growth factor;
(5). changed a subculture in per 3 days, and went down to posterity when cell reached about 80% fusions to 8-9 days, the culture medium that goes down to posterity is the special-purpose serum-free medium of MSC.
6. the preparation method of mescenchymal stem cell injection according to claim 4 is characterized in that the preparation of described placenta mesenchyma stem cell may further comprise the steps:
(1). with the PBS buffer flushing Placenta Hominis that contains 100kU/L penicillin, 100mg/L streptomycin and 50u/ml heparin sodium, along placental edge fetus face amniotic membrane is slowly peeled off;
(2). again wash Placenta Hominis source amniotic membrane, amniotic membrane is cut into 1-5mm
3Fritter;
(3). with the amnion tissue piece DMEM culture medium mixing that contains penicillin and streptomycin, centrifugal 10min under the 700-950g condition;
(4). abandon supernatant, every pipe adds and contains volume ratio 0.25% tryptic DMEM cultivation based on 37 ℃ of digestion 10min, centrifugal 10min under the 700-950g condition;
(5). abandon supernatant, after every pipe adds complete culture medium, centrifugal 10min under the 2200rpm condition; Described complete medium is the MSC serum-free medium that contains 100kU/L penicillin+100mg/L streptomycin;
(6). abandon supernatant, the amnion tissue piece is inoculated in the culture dish, adds complete culture medium, cultivates at incubator; Carry out half amount in per 3 days and change liquid;
(7). the cell attachment growth was arranged to 10-12 days, behind the cell clonal formation, discard piece of tissue, add complete culture medium and cultivate;
(8). merge to 80-90% to 15-17 days cell clonies, carry out passage.
7. the application of mescenchymal stem cell injection according to claim 1 in preparation treatment ulcerative colitis medicine.
8. the application of mescenchymal stem cell injection according to claim 7 in preparation treatment ulcerative colitis medicine, it is characterized in that: the consumption of described mescenchymal stem cell injection is for containing 1 * 10
6-1 * 10
8The mescenchymal stem cell of individual/ml.
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| PCT/CN2013/086838 WO2014075593A1 (en) | 2012-11-14 | 2013-11-11 | Mesenchymal stem cell injection, preparation method thereof, and application thereof in preparing drug for treating ulcerative colitis |
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| WO2014075593A1 (en) * | 2012-11-14 | 2014-05-22 | 贾在美 | Mesenchymal stem cell injection, preparation method thereof, and application thereof in preparing drug for treating ulcerative colitis |
| CN106212443A (en) * | 2016-08-12 | 2016-12-14 | 四川驰鼎盛通生物科技有限公司 | Clinical grade Cell protective solutions and its preparation method and application |
| CN106754668A (en) * | 2016-11-16 | 2017-05-31 | 沈阳细胞治疗工程技术研发中心有限公司 | A kind of stem cell medium and parenteral solution |
| CN106922648A (en) * | 2017-02-22 | 2017-07-07 | 海南新生命干细胞医疗有限公司 | A kind of mescenchymal stem cell cryopreservation solution and preparation method thereof |
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| CN106982821A (en) * | 2017-05-22 | 2017-07-28 | 安徽瑞杰赛尔生物科技有限公司 | Umbilical cord mesenchymal stem cells clinic freezes protection liquid composition and application thereof |
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| CN111603482A (en) * | 2020-05-28 | 2020-09-01 | 高连如 | Mesenchymal stem cell medicine for regulating immune function and preparation method thereof |
| CN112655702A (en) * | 2020-12-31 | 2021-04-16 | 青岛奥克生物开发有限公司 | Solution for umbilical cord mesenchymal stem cells, umbilical cord mesenchymal stem cell preparation, preparation method and application |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10456423B2 (en) | 2016-06-13 | 2019-10-29 | SMART SURGICAL, Inc. | Compositions for biological systems and methods for preparing and using the same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102186338A (en) * | 2008-08-20 | 2011-09-14 | 人类起源公司 | Improved cell composition and methods of making the same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102920734A (en) * | 2012-11-14 | 2013-02-13 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection and preparation method thereof as well as application in preparation of medicine for treating ulcerative colitis |
-
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- 2012-11-14 CN CN2012104570471A patent/CN102920734A/en active Pending
-
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- 2013-11-11 WO PCT/CN2013/086838 patent/WO2014075593A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102186338A (en) * | 2008-08-20 | 2011-09-14 | 人类起源公司 | Improved cell composition and methods of making the same |
Non-Patent Citations (3)
| Title |
|---|
| 《组织工程与重建外科杂志》 20111015 刘静等 "脐带间充质干细胞输注治疗顽固性溃疡性结肠炎" 第258-260页 第7卷, 第5期 * |
| 刘静等: ""脐带间充质干细胞输注治疗顽固性溃疡性结肠炎"", 《组织工程与重建外科杂志》 * |
| 郭葆玉主编: "《生物技术制药》", 30 September 2011, 清华大学出版社 * |
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| CN103421739A (en) * | 2013-05-24 | 2013-12-04 | 北京汉氏联合生物技术有限公司 | Method for separating umbilical cord mesenchymal stem cell effectively |
| CN103421739B (en) * | 2013-05-24 | 2015-09-09 | 北京汉氏联合生物技术有限公司 | A kind of method of high efficiency separation umbilical cord mesenchymal stem cells |
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| CN111603482A (en) * | 2020-05-28 | 2020-09-01 | 高连如 | Mesenchymal stem cell medicine for regulating immune function and preparation method thereof |
| CN112655702A (en) * | 2020-12-31 | 2021-04-16 | 青岛奥克生物开发有限公司 | Solution for umbilical cord mesenchymal stem cells, umbilical cord mesenchymal stem cell preparation, preparation method and application |
| CN114107185A (en) * | 2021-11-01 | 2022-03-01 | 上海市东方医院（同济大学附属东方医院） | Pharmaceutical composition for treating Crohn's disease and preparation method thereof |
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| CN115737699B (en) * | 2022-11-22 | 2024-03-08 | 百欧派(天津)生物技术有限公司 | Stem cell derivative preparation and preparation method and application thereof |
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Also Published As
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| WO2014075593A1 (en) | 2014-05-22 |
| WO2014075593A9 (en) | 2014-07-03 |
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