http://rdf.ncbi.nlm.nih.gov/pubchem/patent/ES-2582369-T3
Outgoing Links
| Predicate | Object |
|---|---|
| assignee | http://rdf.ncbi.nlm.nih.gov/pubchem/patentassignee/MD5_d53b0a261b8b72ec47db98eab5e6b0b7 |
| classificationCPCAdditional | http://rdf.ncbi.nlm.nih.gov/pubchem/patentcpc/C12N2509-00 |
| classificationCPCInventive | http://rdf.ncbi.nlm.nih.gov/pubchem/patentcpc/C12N5-0668 http://rdf.ncbi.nlm.nih.gov/pubchem/patentcpc/C12N5-0605 http://rdf.ncbi.nlm.nih.gov/pubchem/patentcpc/A61P43-00 |
| classificationIPCInventive | http://rdf.ncbi.nlm.nih.gov/pubchem/patentipc/C12N5-073 http://rdf.ncbi.nlm.nih.gov/pubchem/patentipc/C12N5-0775 |
| filingDate | 2008-10-03-04:00^^<http://www.w3.org/2001/XMLSchema#date> |
| grantDate | 2016-09-12-04:00^^<http://www.w3.org/2001/XMLSchema#date> |
| inventor | http://rdf.ncbi.nlm.nih.gov/pubchem/patentinventor/MD5_457cdaefdf3e03c7e6c1ebb69370f4bf http://rdf.ncbi.nlm.nih.gov/pubchem/patentinventor/MD5_c93226a954fa3e2a7a7d9a08bc964240 http://rdf.ncbi.nlm.nih.gov/pubchem/patentinventor/MD5_f6dfe8cae61e49bbda2eb7802b518ea9 http://rdf.ncbi.nlm.nih.gov/pubchem/patentinventor/MD5_2eb20ced5ef53d16d1a08e6453f243a1 http://rdf.ncbi.nlm.nih.gov/pubchem/patentinventor/MD5_68f64f2baddafe204ceee00af569d179 http://rdf.ncbi.nlm.nih.gov/pubchem/patentinventor/MD5_8622ccaab23e4fff39ee977953edfd85 http://rdf.ncbi.nlm.nih.gov/pubchem/patentinventor/MD5_176a384f5455e0f741faa53e5e6b6b2a |
| publicationDate | 2016-09-12-04:00^^<http://www.w3.org/2001/XMLSchema#date> |
| publicationNumber | ES-2582369-T3 |
| titleOfInvention | Optimized and defined method for the isolation and preservation of human umbilical cord precursor cells |
| abstract | Selection method for isolating cells from the matrix of the human umbilical codon characterized in that it comprises the following stages: a) A recovery stage in which the human umbilical cord is transported to the laboratory facilities in a sterile closed container, either dry or submerged in a sterile buffer solution, with or without antibiotics, which preferably contains 186 μg / ml of CaCl2 · 2H2O, 400 μg / ml of KCl, 60 μg / ml of K2HPO4, 200 μg / ml of MgSO4 · 7H2O, 8000 μg / ml NaCl, 350 μg / ml NaHCO3, 90 μg / ml NaH2PO4 · 7H2O, 2000 μg / ml glucose, 10 U / ml penicillin and 10 μg / ml streptomycin; preferably at room temperature if it is processed in a period of 72 h, or between 2 and 8 ° C, preferably at 4 ° C, if it is processed in a period between 48 and 144 h after collection; b) Three stages of washing the umbilical cord with a sterile saline solution, which preferably contains 186 μg / ml of CaCl2,2H2O, 400 μg / ml of KCl, 60 μg / ml of K2HPO4, 200 μg / ml of MgSO4 · 7H2O, 8000 μg / ml NaCl, 350 μg / ml NaHCO3, and 90 μg / ml NaH2PO4 · 7H2O; c) A stage of removal of the external amniotic membrane, where the amniotic membrane is removed with the help of sterile forceps; d) A fractionation stage where the umbilical cord is divided transversely along its longitudinal axis with the help of a scalpel to produce 2.5 cm (2.5 g) fractions; e) A stage of clot removal of the resulting fractions of d), where incisions are made, with the help of a scalpel, at the location where the clots are identified, and where the clots are removed; f) A grouping stage where the resulting fractions of e) meet in groups of 7, which will be processed independently in the following stages; g) A stage of cell dissociation for each group of 7 fractions, resulting from f), carried out in a sterile and sealed flask containing a digestion solution with buffered pH, by the combined action of collagenase II and trypsin, where collagenase II is at a concentration between 0.0375% and 0.075% (weight / volume of total digestion) and trypsin is at a concentration of 0.125% (weight / volume of total digestion); maintaining a constant relationship between tissue mass (grams), the surface area of the bottom of the flask (cm2), digestion volume (ml), and the total volume of the flask (ml), of approximately 1: 2: 2 : 37; and where the flask is incubated under defined conditions of incubation time, temperature, thermal dispersion, ambient humidity and agitation; more specifically, starting from a group of 7 fractions of umbilical cords with approximately 2.5 g each, free of blood clots; using a volume of digestion solution of 35 ml; in a non-ventilated and closed culture flask such as a T175 with a total volume of 650 ml, and a head space during digestion of 615 ml minus the submerged volume of the 7 fractions in digestion; and where the digestion solution consists of, in addition to enzymes, 186 μg / ml of CaCl2 · 2H2O, 400 μg / ml of KCl, 60 μg / ml of K2HPO4, 200 μg / ml of MgSO4 · 7H2O, 8000 μg / ml of NaCl, 350 μg / ml NaHCO3, 90 μg / ml NaH2PO4 · 7H2O, 1000 μg / ml glucose, and 76 μ / ml (0.260 mM) EDTA; maintaining the pH at 6.4 or higher; and where the enzymatic reaction is incubated for a period of 4 h; at a constant temperature of 37 ° C; in a closed dry incubator; with stirring at a constant speed of 100 or 140 oscillations. min-1 (opm); preferably 100 opm, h) A first stage of recovery of dissociated tissue cells, as described in g), which are recovered by static horizontal incubation of the flask when digestion takes place; for a period of 5 to 300 minutes, preferably 30 minutes; at room temperature; followed by transfer of the digestion supernatant by means of pipetting, avoiding the suction of any undigested tissue, to a 50 ml centrifuge tube; which in turn is followed by the removal of all undigested tissue from the digestion flask; to which 35 ml of basal culture medium supplemented with deoxyribonucleases, ribonucleosides, glutamine, antibiotics and 10% bovine fetal serum (FBS) and antibiotics are added; and wherein the cap of the unvented flask was replaced by a filter containing a vented cap; followed by incubation at 37 ° C, in a humidified atmosphere containing 7% CO2; with changes of the culture medium every 72 h to stimulate adhesion and cell growth / multiplication until maximum surface confluence is obtained; i) A second stage of cell recovery, where dissociated tissue cells, as described in g) and contained in the centrifugal supernatant obtained as described in h) are recovered by centrifugation of the 50 ml h centrifuge tube ) at 350 g, for 10 minutes at room temperature; transfer 35 ml of supernatant volume after centrifugation to a static culture flask (T175) with a filter containing a vented cap; add 35 ml of base culture medium supplemented with deoxyribonucleosides, ribonucleosides, glutamine, antibiotics and 10% calf serum (FBS) and antibiotics to the same culture flask. incubate the culture flask at 37 ° C, in a humidified atmosphere containing 7% CO2; and change the culture medium every 72 h to stimulate adhesion and cell growth / multiplication until maximum surface confluence is obtained; j) A third recovery stage where dissociated tissue cells, as described in g) are recovered as a cell agglomerate obtained by centrifuging the digestion supernatant as described in i), resuspended in 2 ml of a solution consisting in bovine fetal serum (FBS), and 10% dimethyl sulfoxide (DMSO), and direct cryopreservation using a controlled speed freezer with a temperature decrease rate of 1 ° C. min-1, up to -80 ° C, in a sterile 2.5 ml cryovial containing 2 ml of cell suspension and 0.5 ml of empty space; k) A cryopreservation stage of the originated cell populations as described in h) and i), after the cell cultures reach the maximum surface confluence, which consists of the direct cryopreservation in the liquid nitrogen vapor phase of a mixture of 0.5 ml of cell suspension and 0.5 ml of a 10% bovine fetal serum solution (FBS) and 10% dimethylsulfoxide (DMSO), so that the final cell density after cryopreservation is approximately 3x106 cell / ml, in a sterile 1.5 ml cryovial, containing the final 1.0 ml of the cell suspension and 0.5 ml of empty space. |
| priorityDate | 2007-10-04-04:00^^<http://www.w3.org/2001/XMLSchema#date> |
| type | http://data.epo.org/linked-data/def/patent/Publication |
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Total number of triples: 170.