CN109453202A - It is a kind of can direct injection application cell preparation and preparation method thereof - Google Patents
It is a kind of can direct injection application cell preparation and preparation method thereof Download PDFInfo
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- CN109453202A CN109453202A CN201811605782.6A CN201811605782A CN109453202A CN 109453202 A CN109453202 A CN 109453202A CN 201811605782 A CN201811605782 A CN 201811605782A CN 109453202 A CN109453202 A CN 109453202A
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- cell
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- cell preparation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/33—Fibroblasts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
Abstract
The present invention provides it is a kind of can direct injection application cell preparation and preparation method thereof.Cell preparation of the present invention, including umbilical cord mesenchymal stem cells, fibroblast, placenta stem-cell, fat stem cell, NK cell, CIK cell, DC cell etc., it is handled by various composition solution, keep manufactured cell preparation bioactivity high and is suitable for human injection.
Description
Technical field
The present invention relates to one kind can direct injection application, and the cell preparation that cell activity is high.It is directed to fill between umbilical cord
The cultural method of matter stem cell, placenta stem-cell, fibroblast, fat stem cell, NK cell, CIK cell, DC cell etc. and
The preparation of its cell preparation.
Background technique
Cell preparation for clinical application can be divided mainly into attached cell preparation and suspension cell preparation two major classes.It is adherent
Cell preparation uses umbilical cord mesenchymal stem cells, mesenchymal stem cell, navel blood stem cell, fibroblast etc. mostly.Patch
Parietal cell plays a significant role in collective's development and organ-tissue update.Wherein umbilical cord mesenchymal stem cells are retrievable thin
Born of the same parents' quantity is more, energetic, convenient for amplification and passage, while again without distribution type, rejection the problems such as, be most potential applicability in clinical practice
Multipotential stem cell can be used as ideal seed cell for injuries of tissues and organs reparation caused by aging and lesion.It is repaired in wound
Multiple later period, fibroblast participate in the reconstruction organized after repairing by secretion clostridiopetidase A.And with the development of research, do thin
Born of the same parents can be used for a variety of diseases such as hepatic failure, cardiovascular disease, infertile, spinal cord injury, nervus retrogression.
Suspension cell preparation mainly has NK cell, CIK cell, DC cell etc..NK cell is the important immunocyte of body,
It is not only related with antitumor, viral infection resisting and immunological regulation, but also hypersensitivity and autoimmunity are participated in some cases
The generation of property disease；DC cell is to be currently known the internal strongest antigen presenting cell of submission function, is trained through external massive amplification
It supports, after tumour antigen sensitization, feeding back can promote the T cell Proliferation, Differentiation of patient to be cytotoxic T cell while swashing to patient
Bone-marrow-derived lymphocyte living starts antibody mediated immunity system comprehensively and generates efficient and special anti-tumor effect；CIK cell is by cell factor
In vitro induction and massive amplification have the cell of tumor killing activity, with powerful anti-tumor activity and non-limiting tumor of killing
Advantage can be used for the tumor patient of any phase.This cells against tumor cells accurately identify killing it is very capable.
The application of immunocyte is patient's treatment after opponent is postoperative or chemicotherapy, it, which can be eliminated, remains small transfer
Lesion prevents tumour cell from spreading and recurring, and can improve immunity of organisms.
The approach that cell is applied to human body has injection infusion, locally injecting, intervention to give the modes such as cell, and injection is transfused this
Kind mode is because the advantages that its is easy to operate, adjusts whole body, and immune state effect is good, is as more commonly used method.Clinical cytology is answered
Infusion is usually required repeatedly with a course for the treatment of, and each Infusion Time combines various aspects situation to provide by expert.
Due to all will affect the use of cell in cell culture medium containing many indefinite substances.Cell leaves culture ring
Starvation can be in behind border, at this moment cell can decompose a certain amount of albumen and carry out maintenance metabolism.And it is needed for human injection
Guarantee the quality of the sample and finished product in cell product production.Need cell characteristics analysis, functional selection, purity analysis and peace
Full property etc. ensures massless problem, and can be further added by other relevant research projects according to the self-characteristic of product.Carefully
Born of the same parents' CHARACTERISTICS IDENTIFICATION includes cell genotype, phenotypic evaluation, differentiation potential, the expression of surface marker, biochemical activity, exogenous
Response and the identification of qualitative and quantitative of expression product of stimulation etc..So injection application cell preparation need to solve more than
Critical issue.
Summary of the invention
The purpose of the present invention is to provide a kind of injectable cell preparations.
Cell preparation of the present invention is after being handled with physiological saline etc. cell, with A liquid, B liquid to cell into
Row processing, be presented on cell will not under starvation, and be used directly for human injection's use.
The isotonic solution such as physiological saline or glucose solution used in the present invention refer to Physiology Experiment or clinically common
The osmotic pressure sodium chloride solution equal with the osmotic pressure of animal or human plasma or glucose solution etc..Referred to using physiological saline
Mass fraction be 0.9% chloride injection solution or 5% glucose solution, it is consistent with the osmotic pressure of tissue, with
It makees solvent, will not be thin to umbilical cord mesenchymal stem cells, placenta stem-cell, fibroblast, fat stem cell, NK cell, CIK
The generations such as born of the same parents, DC cell damage.
Human serum albumin used in the present invention is the protein that content is most in blood plasma, account for Total plasma protein 40%~
60%.It is able to maintain that the constant of plasma colloid osmotic pressure and undertakes certain transportation function.Also have been reported that confirmation in cell preparation
Middle addition albumin can improve the survival rate of cell in the formulation for cells with nutrient, and cell preparation is in low-temperature preservation one
The sign of differentiation is all had no after a month.In a certain range, the additive amount of albumin and the survival rate of cell are positively correlated.But it crosses
High albumin concentration will lead to cell dehydration due to osmotic unbalances and deform, and reduce the survival rate of cell instead.
Cell preparation is made in cell, need to pass through following measurement for clinical application
(1) cell identifies
It is detected by cellular morphology, science of heredity, surface marker or specific gene expression product etc., to different donors and not
The cell of same type carries out comprehensive cell and identifies.
(2) survival rate and growth activity
Sentenced using different activity of cell biology detection methods, such as viable count, cell doubling time, cell cycle
Disconnected cell activity and upgrowth situation.
(3) purity and homogeneity
The detection of cell purity or homogeneity is carried out to preparation by detection cell surface marker etc..
(4) abnormal immune is reacted
Xenogenic origin cell preparation is detected to be proliferated people's total lymphocyte and to different lymphocyte subgroup proliferative capacities
It influences, or the influence to relevant cell factor secretion, to detect the abnormal immune reaction that cell preparation may cause.
(5) oncogenicity
Cell preparation for xenogenic origin or the autogenous cell preparation through external complex operations, must be dynamic by immune deficiency
Tumorigenesis is tested in object, examines the oncogenicity of cell.
(6) biological efficacy is tested
Can by detect cell differentiation potential, induce noble cells structure and physiological function, to the adjusting of immunocyte
Ability, the secretion specific cells factor, the expression functions such as specific gene and albumen, judge cell preparation and treatment-related biology
Validity.To mesenchymal cell, no matter which kind of source, the detection of external multiple types cell differentiation should be carried out, with judgement
The versatility of its cell differentiation.
Cell can be used for the preparation of cell preparation through detecting qualified rear.
It may be directly applied to the cell preparation of human injection, need in the preparation according to current edition " People's Republic of China's medicine
Allusion quotation " in biological products sterility test and detection of mycoplasma regulation, bacterium, fungi and mycoplasma contamination are detected.Foundation
Endotoxin in the current edition Pharmacopoeia of the People's Republic of China detects regulation, and induced by endotoxin is detected.
It can be applied to human injection or part note after last bacterial examination result, the review of operating procedure and label verification etc. are errorless
It penetrates.
The preparation method of attached cell preparation is to cultivate the umbilical cord mesenchymal stem cells, placenta stem-cell, fat to do
The extremely required algebra such as cell, fibroblast；Digestion is cleaned after collecting cell with physiological saline；Cell is mixed with A, B liquid
It is even, cell suspension is made, be made umbilical cord mesenchymal stem cells preparation, placenta stem-cell preparation, fat stem cell preparation, at fiber
Cell preparation etc..
The preparation method of suspension cell preparation is the suspension cells such as the culture NK cell, CIK cell, DC cell；From
The heart collects cell, is cleaned with physiological saline；Cell is uniformly mixed with A, B liquid, cell suspension is made, be made NK cell preparation,
CIK cell preparation, DC cell preparation etc..
The above cell preparation can select suitable cell density to carry out human injection or locally injecting according to practical application.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
Umbilical cord mesenchymal stem cells are obtained using the method for uterus tissue pieces.Culture obtains primary cell, carries out passage training
It supports.With 0.5~1.2 × 106The quantity of/ml is laid on 175cm2Culture bottle in culture to forth generation, change liquid detection every other day.Activation
Cell is cleaned after obtaining the mescenchymal stem cell of activation with physiological saline, and cell is uniformly mixed with A, B liquid, and it is outstanding that cell is made
Umbilical cord mesenchymal stem cells preparation is made in liquid.Quality approval, such as immunogenicity are carried out to final products after completing operation.Including
Identification, potency, purity, impurity, viable count, cell survival rate and detection without bacterium, fungi, mycoplasma, endotoxin, appearance,
PH, osmotic pressure, particulate matter control etc. and the review of operating procedure and label verification etc..It may be directly applied to human body after errorless
Human injection.
Embodiment 2
Fibroblast is obtained using the method for uterus tissue pieces.Culture obtains primary cell, carries out secondary culture.With
0.9~1.5 × 106The quantity of/ml is laid on culture in the culture bottle of 175cm2 and changes liquid detection every other day to the third generation.Active cell,
It is cleaned after obtaining the fibroblast of activation with physiological saline, active cell uses physiology salt after obtaining the fibroblast of activation
Water cleaning, cell is uniformly mixed with A, B liquid, cell suspension is made, fibroblast preparation is made.It completes after operating to final
Product carries out quality approval, such as cellular morphology, viable count, cell survival rate, color, turbidity, particulate matter, visible foreign matters, nothing
The review of bacterium, fungi etc. and operating procedure and label verification etc..After errorless suitable dosage can be selected according to application requirement
And syringe is used for the locally injecting of human body.
Embodiment 3
Various cell preparations be stored in 4 DEG C or under the conditions of 4 DEG C simulation transport, and 0h, 6h, 12h, 18h, for 24 hours,
The time point of 32h extracts the measurement that sample carries out Cell viability and cell quantity.And 0h, 12h and sample for 24 hours is taken to carry out cell
The measurement such as genotype, the identification of phenotype, the expression of surface marker, biochemical activity.
Obtained testing result shows cell genotype, phenotype, the expression of surface marker, biochemical activity of cell etc.
Do not show exception, and for 24 hours when Cell viability 98%, loss cell rate is less than 1%.
Claims (9)
1. a kind of cell preparation, which is characterized in that the cell preparation includes A liquid, B liquid and component C.
2. cell preparation according to claim 1, which is characterized in that A liquid is isotonic solution, such as 0.9% chloride injection
Liquid, glucose solution etc..
3. cell preparation according to claim 1, which is characterized in that B liquid include human serum albumin, mannitol, amino acid,
The nutriments such as electrolyte, microelement, vitamin, injection lecithin.
4. cell preparation according to claim 1, which is characterized in that the component C is umbilical cord mesenchymal stem cells, tire
One of disk stem cell, fibroblast, fat stem cell, NK cell, CIK cell, DC cell etc..
5. cell preparation according to claim 1, which is characterized in that a kind of system of the attached cell preparation of activation
Preparation Method: the steps include: a) to cultivate umbilical cord mesenchymal stem cells described in claim 1, placenta stem-cell, fat stem cell,
The extremely required algebra such as fibroblast；B) cell in collection step a is digested, is cleaned with physiological saline；C) by cell and A, B liquid
Be uniformly mixed, cell suspension is made, be made umbilical cord mesenchymal stem cells preparation, placenta stem-cell preparation, fat stem cell preparation,
Fibroblast preparation etc..
6. cell preparation according to claim 1, which is characterized in that a kind of preparation side of suspension cell preparation
Method: it the steps include: a) suspension cells such as culture NK cell described in claim 1, CIK cell, DC cell；B) step is collected by centrifugation
Cell in rapid a, is cleaned with physiological saline；C) cell is uniformly mixed with A, B liquid, cell suspension is made, NK cell system is made
Agent, CIK cell preparation, DC cell preparation etc..
7. cell preparation described in -6 according to claim 1, which is characterized in that arbitrary cell quantity can be selected according to application.
8. cell preparation is through cellular morphology, viable count, cell survival rate, color, turbidity, particulate matter and visible foreign matters etc., with
And bacterial examination, operating procedure review and label verification etc. it is errorless after can be applied to human injection injection or locally injecting.
9. cell preparation described in -8 according to claim 1, which is characterized in that the Cell viability of cell preparation 98% or more and
The quality for not changing cell preparation for 24 hours can be saved at 4 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811605782.6A CN109453202A (en) | 2018-12-21 | 2018-12-21 | It is a kind of can direct injection application cell preparation and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811605782.6A CN109453202A (en) | 2018-12-21 | 2018-12-21 | It is a kind of can direct injection application cell preparation and preparation method thereof |
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| CN109453202A true CN109453202A (en) | 2019-03-12 |
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| CN201811605782.6A Withdrawn CN109453202A (en) | 2018-12-21 | 2018-12-21 | It is a kind of can direct injection application cell preparation and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110237095A (en) * | 2019-05-28 | 2019-09-17 | 武汉汉密顿生物科技股份有限公司 | For treating stem cell injection liquid and its preparation and the application of cartilage defect of osteoarthritis |
| WO2023016510A1 (en) * | 2021-08-13 | 2023-02-16 | 无锡赛比曼生物科技有限公司 | Preservation solution for low-temperature preservation of adipose-derived mesenchymal stem cells |
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| CN102008507A (en) * | 2010-11-21 | 2011-04-13 | 天津和泽干细胞科技有限公司 | Human umbilical cord mesenchymal stem cell (HUMSC) anti-hepatic fibrosis injection and preparation method thereof |
| CN106237313A (en) * | 2016-09-30 | 2016-12-21 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord mesenchymal stem cells injection 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 |
| CN106924285A (en) * | 2017-03-17 | 2017-07-07 | 广州赛莱拉干细胞科技股份有限公司 | A kind of placenta mesenchyma stem cell parenteral solution and its preparation method and application |
| CN107090431A (en) * | 2017-03-31 | 2017-08-25 | 北京恒峰铭成生物科技有限公司 | A kind of active mesenchymal stem cell injection for being used to be transfused diabetes |
-
2018
- 2018-12-21 CN CN201811605782.6A patent/CN109453202A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008507A (en) * | 2010-11-21 | 2011-04-13 | 天津和泽干细胞科技有限公司 | Human umbilical cord mesenchymal stem cell (HUMSC) anti-hepatic fibrosis injection and preparation method thereof |
| CN106237313A (en) * | 2016-09-30 | 2016-12-21 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord mesenchymal stem cells injection 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 |
| CN106924285A (en) * | 2017-03-17 | 2017-07-07 | 广州赛莱拉干细胞科技股份有限公司 | A kind of placenta mesenchyma stem cell parenteral solution and its preparation method and application |
| CN107090431A (en) * | 2017-03-31 | 2017-08-25 | 北京恒峰铭成生物科技有限公司 | A kind of active mesenchymal stem cell injection for being used to be transfused diabetes |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110237095A (en) * | 2019-05-28 | 2019-09-17 | 武汉汉密顿生物科技股份有限公司 | For treating stem cell injection liquid and its preparation and the application of cartilage defect of osteoarthritis |
| WO2023016510A1 (en) * | 2021-08-13 | 2023-02-16 | 无锡赛比曼生物科技有限公司 | Preservation solution for low-temperature preservation of adipose-derived mesenchymal stem cells |
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Application publication date: 20190312 |