CN108670946A - Treat Cellular gels preparation of articular cartilage damage and application thereof and the active gel solution of holding freeze-stored cell used - Google Patents
Treat Cellular gels preparation of articular cartilage damage and application thereof and the active gel solution of holding freeze-stored cell used Download PDFInfo
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- 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
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
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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
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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
- A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
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- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
-
- 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
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
Abstract
The invention discloses a kind of Cellular gels preparations for treating articular cartilage damage and application thereof and the active gel solution of holding freeze-stored cell used, the formula of Cellular gels preparation is by percent by volume, gel solution is 50%~95%, compound electrolyte cell re-suspension liquid is 5%~50%, contains 0.5 × 10 in every milliliter of Cellular gels preparation5~2 × 107A cell.Gel solution includes following quality and volume constituents：1~50mg of Polymer material stabilizer, 0.1~1mg of chondroitin sulfate, 5~50mg of human serum albumin, glycerine 1~30ul of 30~150ul, DMSO, Multiple electrolytes injection or amino acid injection or normal saline solution are 700~900ul.Gel preparation good biocompatibility can directly carry out low temperature long-term preservation and maintain higher cell activity, not need traditional liquid nitrogen cryopreservation, and the Cellular gels preparation after recovery still is able to maintain the biological characteristics of cell, function and its survival rate is high in articular cavity.It can be used for the treatment use of articular cartilage damage.
Description
Technical field
The present invention relates to biotechnology and biomedicine field, more particularly to a kind of cell for treating articular cartilage damage is solidifying
Glue preparation and application thereof and the active gel solution of holding freeze-stored cell used.
Background technology
Articular cartilage defect is very common in China's clinic at present, the cartilage damage patient increased newly every year in China according to statistics
Nearly 10,000,000.Due to articular cartilage surface tissue impassivity, without blood vessel and without lymphatic return, therefore the cartilaginous tissue damaged is difficult to
Self-regeneration.Clinically being used for repairing articular cartilage damage method at present has：Joint fusion, joint debridement art, arthrectomy at
Type art, artificial joint replacement, self or homogenous cartilage transplantation, the Autologous Chondrocyte transplantation of matrix induction, Yi Jiji
Because of therapy and stem cell therapy etc..Although this method energy respite pain, late result is unsatisfactory, wherein operation is repaired
Cartilage mode can not substitute the cartilaginous tissue with full biological activity function completely, serious function of joint to be caused to lose.
Hyaluronic acid (HA) is the acid mucopolysaccharide in animal body, because having good biocompatibility, biological degradability,
Saving cortilage cartilage is mitigated or eliminated arthralgia, inhibits cartilage degeneration characteristic, can be used for studying syringeability group weaver
The structure of journey cartilage, and by FDA approvals for clinic.
Chitosan be by chitin through deacetylation from, have good biocompatibility, biodegradable absorb
Property, hydrophily and non-immunogenicity.Meanwhile the chitosan being made of gucosamine and n- acetylglucosamines class in structure
It is similar to glucose aminoglucan, is present in the extracellular matrix of cartilaginous tissue.Hydrogel based on chitosan, which has, maintains cell
Activity there is protection and supporting function, the protective agent of cartilaginous tissue to become the ideal chose of repair of cartilage cell.
Collagen is as the main component part of articular cartilage, the polymer being made of a variety of amino acid.With good raw
Object compatibility, degradability, water absorbing and retaining properties are completely degraded absorption in vivo, nontoxic to human body nonantigenic.Meanwhile collagen egg
White reticular structure is the frame of articular cartilage, can inhibit cartilage degeneration, reduces friction of the bone between bone, therefore collagen is one
The ideal cartilage damage repair materials of kind.
Constituent of the chondroitin sulfate as cartilage, it to maintain cellular environment relatively stablize and normal physiological function
It plays an important role, and plays the role of anti-inflammatory, accelerating wound healing and antitumor etc..What is more important chondroitin sulfate
Conveyance conduit is can be used as, oxygen and nutrient delivery to joint, while carbon dioxide and waste are excluded, to promote
The effect of regenerating bone or cartilage.Increase in addition, the addition of chondroitin sulfate contributes to the fiber of cartilage cell to link protein expression, the egg
It is white generally to come across in chondrogenetic early stage and ripe articular cartilage.
Mescenchymal stem cell (MSCs) because with powerful self-renewing, proliferative capacity and differentiation potential, low immunogenicity,
Histocompatbility is good, discharges the advantages that secretion factor and is concerned.It can retain its stem cell properties in amplification procedure simultaneously,
Promote the differentiation of cartilage cell.These features make MSCs be increasingly becoming the ideal seed cell for repairing cartilage defect.
Cartilage cell is the important component of articular cartilage, by secreting some cells and growth factor come soft to joint
Bone tissue is regulated and controled, and the cell plays an important role in cartilage metabolic processes.At the same time, self soft
Osteocyte transplantation is the main path of current clinical treatment articular cartilage damage.
Currently, South Korea has been approved by the mescenchymal stem cell drug for treating articular cartilage damageListing.But it is to mix mesenchymal stem cells in umbilical cord blood with hyaluronic acid using preceding, is then made again
With.This method can increase the risk of cell contamination, and need stringent operating process.
Invention content
The technical problem to be solved by the invention is to provide a kind of Cellular gels preparation for treating articular cartilage damage and
Its purposes and the active gel solution of holding freeze-stored cell used, can be in all matter using preceding completion including sterile
Amount detection, can use after defrosting, use simplicity immediately.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention：A kind of active gel of holding freeze-stored cell
Solution, including following quality and volume constituents：1~50mg of Polymer material stabilizer, 0.1~1mg of chondroitin sulfate, people's blood are white
5~50mg of albumen, glycerine 1~30ul of 30~150ul, DMSO, Multiple electrolytes injection or amino acid injection or life
Manage 700~900ul of brine injections.
The Polymer material stabilizer is the combination of one or more of Sodium Hyaluronate, chitosan, collagen.
The Polymer material stabilizer, chondroitin sulfate, DMSO, glycerine, human serum albumin, compound electrolyte injection
Liquid, amino acid injection, normal saline solution are all pharmaceutical grade supplementary materials, and wherein normal saline solution mass percent is dense
Degree is concentration 0.9%.
A kind of Cellular gels system of the treatment articular cartilage damage containing the above-mentioned active gel solution of holding freeze-stored cell
Agent, which is characterized in that press percent by volume, gel solution be 50%~95%, compound electrolyte cell re-suspension liquid be 5%~
50%, 0.5 × 10 is contained in every milliliter of Cellular gels preparation5~2 × 107A cell.
The cell is the mixing of one or both of mescenchymal stem cell and cartilage cell.
The source for mesenchymal stem cells is in marrow, umbilical cord, bleeding of the umbilicus, placenta, amnion or adipose tissue.
Above-mentioned Cellular gels preparation is preparing the application in treating articular cartilage damage drug.
The articular cartilage damage includes that traumatic arthritis, degenerative osteoarthritis, rheumatoid arthritis, tendon are repaiied
Multiple art, meniscus displacement technique.
The beneficial effects of the invention are as follows：The gel preparation good biocompatibility can directly carry out low temperature long-term preservation and dimension
Higher cell activity (- 80 DEG C) is held, preparation manipulation is easy, and 80% or more the activity that can maintain cell for 7 days is stored at -20 DEG C,
Do not need traditional liquid nitrogen cryopreservation, the Cellular gels preparation after recovery still be able to maintain the biological characteristics of cell, function and its
Survival rate is high in articular cavity.It can be used for the treatment use of articular cartilage damage.
Description of the drawings
Fig. 1 is umbilical cord mesenchymal stem cells of the present invention in Sodium Hyaluronate/collagen/chondroitin sulfate (1:1:0.1)
Survival rate test figure in gel
Fig. 2 is umbilical cord mesenchymal stem cells of the present invention in Sodium Hyaluronate/collagen/chondroitin sulfate (48:2:1)
Survival rate test figure in gel.
Fig. 3 is cartilage cell of the present invention in chitosan/chondroitin sulfate (1:0.1) the survival rate test figure in gel.
Fig. 4 is cartilage cell of the present invention in chitosan/chondroitin sulfate (50:1) the survival rate test figure in gel.
Fig. 5 is Sodium Hyaluronate/collagen/chondroitin sulfate (25 after Long-term Cryopreservation of the present invention:25:1) gel system
The motility rate curve graph (18 months) of cartilage cell in agent
Fig. 6 is chitosan/collagen/chondroitin sulfate (48 after Long-term Cryopreservation of the present invention:2:1) navel in gel preparation
Motility rate curve graph (18 months) with mescenchymal stem cell
Fig. 7 is Sodium Hyaluronate/collagen/chondroitin sulfate (25 containing umbilical cord mesenchymal stem cells:25:1) it coagulates
Therapeutic effect figure of the glue preparation to new zealand rabbit cartilage defect.
Fig. 8 is Sodium Hyaluronate/collagen/chondroitin sulfate (25 containing umbilical cord mesenchymal stem cells:25:1) it coagulates
Glue preparation is to the arthritic therapeutic effect figure of rat bone.
Specific implementation mode
The present invention is further described with specific embodiment below in conjunction with the accompanying drawings, and specific embodiment is construed as
By way of example only, it is not restrictive, protection scope of the present invention cannot be limited with following illustrations.
A kind of active gel solution of holding freeze-stored cell of the present invention, including following quality and volume constituents：Macromolecule
Material settling out 1~50mg of agent, 0.1~1mg of chondroitin sulfate, 5~50mg of human serum albumin, glycerine 30~150ul, DMSO
1~30ul, Multiple electrolytes injection or amino acid injection or 700~900ul of normal saline solution.
The Polymer material stabilizer is the combination of one or more of Sodium Hyaluronate, chitosan, collagen.
The Polymer material stabilizer, chondroitin sulfate, DMSO, glycerine, human serum albumin, compound electrolyte injection
Liquid, amino acid injection, normal saline solution are all pharmaceutical grade supplementary materials, and wherein normal saline solution mass percent is dense
Degree is concentration 0.9%.
A kind of Cellular gels system of the treatment articular cartilage damage containing the above-mentioned active gel solution of holding freeze-stored cell
Agent, which is characterized in that press percent by volume, gel solution be 50%~95%, compound electrolyte cell re-suspension liquid be 5%~
50%, 0.5 × 10 is contained in every milliliter of Cellular gels preparation5~2 × 107A cell.
The cell is the mixing of one or both of mescenchymal stem cell and cartilage cell.
The source for mesenchymal stem cells is in marrow, umbilical cord, bleeding of the umbilicus, placenta, amnion or adipose tissue.
Above-mentioned Cellular gels preparation is preparing the application in treating articular cartilage damage drug.
The articular cartilage damage includes that traumatic arthritis, degenerative osteoarthritis, rheumatoid arthritis, tendon are repaiied
Multiple art, meniscus displacement technique.
Specifically, cell, that is, the mescenchymal stem cell and/or cartilage cell need by tissue separation and culture, then
It is obtained through passage.
The preparation method with gel preparation containing mescenchymal stem cell and/or cartilage cell, is as follows：
1. the preparation of gel solution：
It is according to above-mentioned offer proportioning, injection stage or pharmaceutical grade supplementary material, glycerine, human serum albumin, DMSO, sulfuric acid is soft
Ossein, is dissolved in Multiple electrolytes injection and/or amino acid injection, normal saline solution are supplied, through be sufficiently stirred mixing,
For use.
2. acquired cell：
Cell needed for being chosen from cell bank is cultivated using the DMEM culture mediums for containing 10% fetal calf serum as complete
Base, cell is in 37 DEG C, 5%CO2Under conditions of cultivated, it is clear with phosphate buffer when cell growth is to 85% or so
Cell is washed, 0.25% trypsase is digested, and then stops its digestion with complete medium, and the cell suspension of acquisition carries out
1500 × g, 5min are centrifuged, and abandon supernatant, then cell is resuspended with 100~300ul Multiple electrolytes injections, and adjustment is thin
Born of the same parents' concentration is to 5 × 105~6 × 107A/milliliter is spare.
3. the preparation of Cellular gels preparation：
The cell suspension prepared is injected into the pre-charge injector containing gel solution, is sufficiently mixed uniformly, directly
It freezes for use in -80 DEG C of refrigerators.
Embodiment 1：Umbilical cord mesenchymal stem cells are in Sodium Hyaluronate/collagen/chondroitin sulfate (1:1:0.1) it coagulates
Survival rate test in glue
Sterile hyaluronic acid sodium (10mg), collagen (10mg) and chondroitin sulfate (1mg) are combined into blended powder
It is dissolved in 8.49ml phosphate solutions, after room temperature is swollen 24 hours, adds 1.5ml glycerine, 50mg human serum albumins,
10ul DMSO, are sufficiently stirred mixing, the high molecular material gel solution being configured to.It is for use after standing, take 900ul gel solutions
100ul is added and contains 5 × 105The compound electrolyte re-suspension liquid of/cells umbilical cord mesenchymal stem cells is uniformly mixed, is made
Bright matter acid sodium/chondroitin sulfate/collagen gel preparation.It directly freezes in -80 DEG C of refrigerators, after 0,7,15 day, each group is each
Take 3 progress Cell viability detections.By experimental result as it can be seen that the gel preparation has good cell compatibility, that is, maintain navel
Activity (Fig. 1) with mescenchymal stem cell
Embodiment 2：Umbilical cord mesenchymal stem cells are in Sodium Hyaluronate/collagen/chondroitin sulfate gel (48:2:1)
In survival rate test
Sterile hyaluronic acid sodium (480mg), collagen (20mg) and chondroitin sulfate (10mg) are combined into blending powder
End is dissolved in 9.4ml amino acid injections, after room temperature is swollen 24 hours, adds 300ul glycerine, 5mg human serum albumins,
300ul DMSO, are sufficiently stirred mixing, the high molecular material gel solution being configured to.It is for use after standing, take 900ul gel solutions
100ul is added and contains 5 × 105The compound electrolyte re-suspension liquid of/cells umbilical cord mesenchymal stem cells is uniformly mixed, is made
Bright matter acid sodium/collagen/chondroitin sulfate gel preparation.It directly freezes in -80 DEG C of refrigerators, after 0,7,15 day, each group is each
Take 3 progress Cell viability detections.By experimental result as it can be seen that the gel preparation has good cell compatibility, that is, maintain navel
Activity (Fig. 2) with mescenchymal stem cell
Embodiment 3：Cartilage cell is in chitosan/chondroitin sulfate (1:0.1) survival rate test in gel
Sterile chitosan (10mg) and chondroitin sulfate (1mg) are combined into blended powder and are dissolved in 8.49m physiological saline note
(normal saline solution mass percent concentration is concentration 0.9%) is penetrated in liquid, after room temperature is swollen 24 hours, adds 1.5ml
Glycerine, 50mg human serum albumins, 10ul DMSO are sufficiently stirred mixing, the high molecular material gel solution being configured to.It stands
It is for use afterwards, it takes 700ml gel solutions that 300ul is added and contains 2 × 107The compound electrolyte re-suspension liquid of/cells cartilage cell is mixed
It closes uniformly, chitosan/chondroitin sulfate gel preparation is made.It directly freezes in -80 DEG C of refrigerators, after 0,7,15 day, each group is each
Take 3 progress Cell viability detections.By experimental result as it can be seen that the gel preparation has good cell compatibility, that is, maintain soft
The activity (Fig. 3) of osteocyte
Embodiment 4：Cartilage cell is in chitosan/chondroitin sulfate (50:1) survival rate test in gel
Sterile chitosan (500mg) and chondroitin sulfate (10mg) are combined into blended powder, and to be dissolved in 9.4ml phosphate molten
In liquid, after room temperature is swollen 24 hours, 300ul glycerine, 5mg human serum albumins are added, 300ul DMSO are sufficiently stirred mixed
High molecular material gel solution that is even, being configured to.It is for use after standing, it takes 700ul gel solutions that 300ul is added and contains 2 × 107/
The compound electrolyte re-suspension liquid of cells cartilage cell is uniformly mixed, chitosan/chondroitin sulfate gel preparation is made.Directly freeze
It is stored in -80 DEG C of refrigerators, after 0,7,15 day, each group respectively takes 3 progress Cell viability detections.By experimental result as it can be seen that the gel
Preparation has good cell compatibility, that is, maintains the activity (Fig. 4) of cartilage cell
Embodiment 5：After Long-term Cryopreservation, Sodium Hyaluronate/collagen/chondroitin sulfate (25:25:1) in gel preparation
The motility rate (18 months) of cartilage cell
Sterile hyaluronic acid sodium (1.25g), collagen (1.25g) and chondroitin sulfate (50mg) are combined into blending powder
End is dissolved in 42.45ml phosphate solutions, after room temperature is swollen 24 hours, adds 7.5ml glycerine, 2.5g human serum albumins,
50ul DMSO, are sufficiently stirred mixing, the high molecular material gel solution being configured to.It is for use after standing, take 800ul gel solutions
200ul is added and contains 1 × 107The compound electrolyte re-suspension liquid of/cells cartilage cell, be uniformly mixed, be made Sodium Hyaluronate/
Collagen/chondroitin sulfate gel preparation.0th, 1,3,6,15,18 month, 3 progress Cell viability detections are respectively taken, and examine
Survey its phenotype and differentiation capability.It is obtained by experimental result, the cartilage cell of gel preparation load can protect -80 DEG C long-term
It deposits to 18 months, it is (Fig. 5) with good stability.
Embodiment 6：After Long-term Cryopreservation, chitosan/collagen/chondroitin sulfate (48:2:1) in gel preparation between umbilical cord
The motility rate (18 months) of mesenchymal stem cells
It is molten that sterile chitosan (2.4g), collagen (100mg) and chondroitin sulfate (50mg) are combined into blended powder
In 47ml phosphate solutions, after room temperature is swollen 24 hours, 1.5ml glycerine, 250mg human serum albumins, 1.5ml are added
DMSO is sufficiently stirred mixing, the high molecular material gel solution being configured to.It is for use after standing, take 800ul gel solutions to be added
200ul contains 1 × 107The compound electrolyte re-suspension liquid of/cells umbilical cord mesenchymal stem cells cells is uniformly mixed, and it is poly- that shell is made
Sugar/collagen/chondroitin sulfate gel preparation.0th, 1,3,6,15,18 month, 3 progress Cell viability detections are taken, and examine
Survey its phenotype and differentiation capability.It is obtained by experimental result, the umbilical cord mesenchymal stem cells of gel preparation load can be -80
DEG C long-term preservation is (Fig. 6) with good stability to 18 months.
Embodiment 7：Sodium Hyaluronate/collagen/chondroitin sulfate (25 containing umbilical cord mesenchymal stem cells:25:
1) therapeutic effect of the gel preparation to new zealand rabbit cartilage defect
40 healthy New Zealand White Rabbit are taken, male and female are unlimited, weight (2.0~2.5kg), random point 4 groups (every group each 10
Only) i.e.：Positive controls-physiological saline group (after processing), negative control group-untreated fish group, treatment group -1：Umbilical cord mesenchyma
Stem cell group, treatment group -2：Cellular gels preparation group.The proportioning provided according to above-described embodiment 5 prepares gel solution, takes
800ul gel solutions are added 200ul and contain 1 × 107The compound electrolyte re-suspension liquid of/cell umbilical cord mesenchymal stem cells, mixing
Uniformly, Sodium Hyaluronate/collagen/chondroitin sulfate gel preparation is made.Animal model：Use outer diameter for the point of 2.0mm
Needle head is slowly firmly rotated in femoral bone pulley central part, until an annulus pitfall occurs in cartilage surface defect, in striking off
It entreats all cartilaginous tissues to subchondral bone calcified layer to expose, completes structure Full-thickness chondral defects model.Treatment：To positive controls
The physiological saline of intraarticular injection 200ul contains 2 × 10 to -1 intraarticular injection for the treatment of group6/ cell umbilical cord mesenchymas are dry
Cell compound electrolyte re-suspension liquid 200ul, to -2 intraarticular injection 200ul Cellular gels preparations for the treatment of group.It is carried out at 12 weeks
Defect cartilaginous tissue repairs ICRS Macroscopic scores.(standards of grading are referring to table -1；Appraisal result is shown in Fig. 7)
1 cartilage damage of table repairs ICRS points-scoring systems
Control group and the defect cartilaginous tissue for the treatment of group are repaired ICRS Macroscopic score results and are shown (Fig. 7)：At 12 weeks, treatment
- 1, -2 appraisal result of group is apparently higher than positive controls i.e. physiological saline group (P<0.05)；Cellular gels preparation group (treatment group-
2) ICRS appraisal results are higher than umbilical cord mesenchymal stem cells group (treatment group -1), and with negative control group (untreated fish group) without aobvious
Write sex differernce (P>0.05).I grades of the former defect depth shallower about ICRS after Cellular gels are treated, surface is smooth.Show articular cavity
Interior injection Cellular gels preparation can be obviously improved and repair the injury tissue of cartilage surface.
Embodiment 8：Sodium Hyaluronate/collagen/chondroitin sulfate (25 containing umbilical cord mesenchymal stem cells:25:
1) gel preparation is to the arthritic therapeutic effect of rat bone
40 healthy SD rats are taken, divide 4 groups (every group each 10) i.e. at random：Positive controls-physiological saline group (place
After reason), negative control group-untreated fish group, treatment group -1：Umbilical cord mesenchymal stem cells group, treatment group -2：Cellular gels preparation
Group.The proportioning provided according to above-described embodiment 5 prepares gel solution, takes 800ul gel solutions that 200ul is added and contains 1 × 107/
The compound electrolyte re-suspension liquid of cell umbilical cord mesenchymal stem cells is uniformly mixed, Sodium Hyaluronate/collagen/sulfuric acid is made
Chondroitin gel preparation.Joint cavity injection sodium iodoacetate solution builds osteoarthritis.Cheng Mo after 4 weeks, to positive controls
The physiological saline of intraarticular injection 100ul contains 1 × 10 to -1 intraarticular injection for the treatment of group6/ cell umbilical cord mesenchymas
Stem cell compound electrolyte re-suspension liquid 100ul, to -2 intraarticular injection 100ul Cellular gels preparations for the treatment of group.It is right after 12 weeks
Cartilage surface is substantially scored.(standards of grading are referring to table -2；Appraisal result is shown in Fig. 8)
2 cartilage surface of table substantially points-scoring system
- 1, -2 cartilage surface of 12 Zhou Hou treatment groups, which substantially scores, is significantly lower than positive controls (P<0.05), i.e., positive right
Occur according to group (after physiological saline group-processing) articular surface rotten to the corn and apparent with surrounding tissue boundary.Cellular gels preparation group is wanted
There is chondroid tissue reparation in slightly above cell therapy group at cartilage defect, surface is completely smooth, with negative control group
(untreated fish group) there was no significant difference (P>0.05).The result shows that intraarticular injection Cellular gels preparation can be obviously improved by closing
Cartilage joint surface damage (Fig. 8) caused by section inflammation.
In conclusion present disclosure is not limited in the above embodiments, the knowledgeable people in same area can
Can propose other embodiments easily within the technological guidance's thought of the present invention, but this embodiment is included in this hair
Within the scope of bright.
Claims (8)
1. a kind of active gel solution of holding freeze-stored cell, which is characterized in that including following quality and volume constituents：Macromolecule
Material settling out 1~50mg of agent, 0.1~1mg of chondroitin sulfate, 5~50mg of human serum albumin, glycerine 30~150ul, DMSO 1
~30ul, Multiple electrolytes injection or amino acid injection or 700~900ul of normal saline solution.
2. keeping the active gel solution of freeze-stored cell according to claim 1, which is characterized in that the high molecular material is steady
Determine the combination that agent is one or more of Sodium Hyaluronate, chitosan, collagen.
3. keeping the active gel solution of freeze-stored cell according to claim 1, which is characterized in that the high molecular material is steady
Determine agent, chondroitin sulfate, DMSO, glycerine, human serum albumin, Multiple electrolytes injection, amino acid injection, physiological saline
Injection is all pharmaceutical grade supplementary material, and wherein normal saline solution mass percent concentration is concentration 0.9%.
4. a kind of containing any one of the claim 1-3 treatment articular cartilage damages for keeping the active gel solution of freeze-stored cell
The Cellular gels preparation of wound, which is characterized in that press percent by volume, gel solution is 50%~95%, compound electrolyte cell
Re-suspension liquid is 5%~50%, contains 0.5 × 10 in every milliliter of Cellular gels preparation5~2 × 107A cell.
5. treating the Cellular gels preparation of articular cartilage damage according to claim 4, which is characterized in that between the cell is
The mixing of one or both of mesenchymal stem cells and cartilage cell.
6. treating the Cellular gels preparation of articular cartilage damage according to claim 5, which is characterized in that the mesenchyma is dry
Cell origin is in marrow, umbilical cord, bleeding of the umbilicus, placenta, amnion or adipose tissue.
7. Cellular gels preparation is preparing the application in treating articular cartilage damage drug as described in claim 4-6 is any.
8. Cellular gels preparation is preparing the application in treating articular cartilage damage drug, feature according to claim 7
It is, the articular cartilage damage includes traumatic arthritis, degenerative osteoarthritis, rheumatoid arthritis, tendon repair
Art, meniscus displacement technique.
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