Foetal polymix of mesenchymal stem cells under hypoxic conditions for the treatment of clinical disorders and diseases

Abstract

A method for isolation, enrichment and co-culture of foetal polymix involving two or more components of mesenchymal stem cells derived from, but not limited to, placenta, amnion, amniotic fluid, chorion and umbilical cord and/or other products of conception under hypoxic or otherwise and/or normoxic/general conditions for treatment of a plurality of disorders ranging from congenital to degenerative to developmental to malignant disorders and diseases prior to therapeutic administration.

1 - 25 . (canceled) 
     
     
         26 . A method making a foetal polymix composition, the method comprising:
 isolating mesenchymal stem cells (MSCs) from two separate sources, wherein the two sources are placenta and umbilical cord tissue;   enriching the MSCs from each source separately; and   co-culturing the enriched MSCs from each source together in a culture medium under hypoxic conditions, wherein the hypoxic conditions comprise incubation in a carbon dioxide incubator at 37° C. with 1% oxygen concentration, and with a remaining concentration containing carbon dioxide and nitrogen,   wherein the MSCs from placenta are isolated and enriched by a process comprising of:
 collecting a chorionic trophoblast from the placenta; 
 washing the chorionic trophoblast with a sterile phosphate buffer solution (PBS); 
 selecting an area from the chorionic trophoblast that contains chorionic wherein the area is minced into a plurality of pieces; 
 adding collagenase and dispase to a container containing minced chorionic trophoblast for digestion; 
 incubating the container; 
 adding fetal bovine serum to the container to inhibit activity of collagenase and dispase; 
 filtering a mixture in the container; centrifuging a filtrate from the mixture to obtain a pellet; 
 re-suspending the pellet in another PBS to obtain a re-suspended pellet layer; 
 adding the re-suspended pellet layer on top of a ficoU density gradient medium to obtain a mixture; 
 centrifuging the mixture to obtain cells from a middle band; 
 washing the cells with PBS to obtain mesenchymal stem cells; 
 re-suspending the mesenchymal stem cells in Dulbecco's Modified Eagle's Medium (DMEM) for culturing; 
 seeding isolated mesenchymal stem cells from DMEM into a first culture vessel; and 
 incubating the seeded isolated mesenchymal stem cells until the seeded isolated mesenchymal stem cells reach 70-80% confluence, 
   wherein the MSCs from an umbilical cord are isolated and enriched by a process comprising of:
 subjecting a processed umbilical cord to an enzymatic digestion with collagenase and hyaluronidase to form a mixture; 
 incubating the mixture containing the processed umbilical cord, collagenase, and hyaluronidase with gentle agitation; 
 adding trypsin to the agitated mixture and then incubating to obtain a digested suspension; 
 diluting the digested suspension with phosphate buffer solution (PBS) to obtain a diluted suspension; 
 filtering the diluted suspension to obtain a single cell suspension; 
 centrifuging the single cell suspension to obtain a pellet; 
 re-suspending the pellet in a washing solution comprising albumin, hydroxyethyl starch (HES), and citrate-phosphate-dextrose-adenine anticoagulant; 
 isolating cells containing MSCs from the washing solution; 
 seeding isolated MSCs with DMEM into a second culture vessel; and 
 incubating the seeded isolated MSCs until the seeded isolated MSCs reach 70-80% confluence. 
   
     
     
         27 . The method of  claim 26 , further comprising wherein the foetal polymix composition is formulated into an injectable composition. 
     
     
         28 . The method of  claim 26 , further comprising wherein the foetal polymix composition is formulated to be suitable for administering via an intravenous route, an intrathecal route, an intra-articular route, an intramuscular route, a derma roller, a mesogun, a local route, a periorbital route, an intravitreal route, or a retrobular route. 
     
     
         29 . The method of  claim 26 , further comprising wherein the foetal polymix composition is formulated to be suitable for treating disorders and diseases. 
     
     
         30 . A method making a foetal polymix composition, the method comprising:
 isolating mesenchymal stem cells (MSCs) from two separate sources, wherein the two sources are placenta and umbilical cord tissue;   enriching the MSCs from each source separately; and   co-culturing the enriched MSCs from each source together in a culture medium under hypoxic conditions, wherein the hypoxic conditions comprise incubation in a carbon dioxide incubator at 37° C. with 1% oxygen concentration, and with a remaining concentration containing carbon dioxide and nitrogen,   wherein the MSCs from placenta are isolated and enriched by a process comprising of:
 collecting a fetal decidua's placental tissue from placenta; 
 washing the fetal decidua's placental tissue with a phosphate buffer solution (PBS); 
 digesting the fetal decidua's placental tissue with collagenase; 
 separating cells by centrifuging a mixture of the fetal decidua's placental tissue and collagenase over a Ficoll-Hypaque separation medium to obtain separated cells; 
 re-suspending the separated cells for culture in L-Dulbecco's Modified Eagle's Medium (LDMEM) supplemented with fetal bovine serum, basic fibroblast growth factor, L-glutamine, and at least one antibiotic; 
 seeding cells from the LDMEM into a first culture vessel; 
 removing non-adherent cells from the first culture vessel; 
 harvesting cells from the first culture vessel by adding trypsin and ethylenediaminetetraacetic acid (EDTA); and 
 passaging the cells, 
   wherein the MSCs from an umbilical cord are isolated and enriched by a process comprising of:
 subjecting a processed umbilical cord to an enzymatic digestion with collagenase and hyaluronidase to form a mixture; 
 incubating the mixture containing the processed umbilical cord, collagenase, and hyaluronidase with gentle agitation; 
 adding trypsin to the agitated mixture and then incubating to obtain a digested suspension; 
 diluting the digested suspension with phosphate buffer solution (PBS) to obtain a diluted suspension; 
 filtering the diluted suspension to obtain a single cell suspension; 
 centrifuging the single cell suspension to obtain a pellet; 
 re-suspending the pellet in a washing solution comprising albumin, hydroxyethyl starch (HES), and citrate-phosphate-dextrose-adenine anticoagulant; 
 isolating cells containing MSCs from the washing solution; 
 seeding isolated MSCs with DMEM into a second culture vessel; and 
 incubating the seeded isolated MSCs until the seeded isolated MSCs reach 70-80% confluence. 
   
     
     
         31 . The method of  claim 30 , further comprising wherein the foetal polymix composition is formulated into an injectable composition. 
     
     
         32 . The method of  claim 30 , further comprising wherein the foetal polymix composition is formulated to be suitable for administering via an intravenous route, an intrathecal route, an intra-articular route, an intramuscular route, a derma roller, a mesogun, a local route, a periorbital route, an intravitreal route, or a retrobular route. 
     
     
         33 . The method of  claim 30 , further comprising wherein the foetal polymix composition is formulated to be suitable for treating disorders and diseases. 
     
     
         34 . A method making a foetal polymix composition, the method comprising:
 isolating mesenchymal stem cells (MSCs) from two separate sources, wherein the two sources are placenta and umbilical cord tissue;   enriching the MSCs from each source separately; and   co-culturing the enriched MSCs from each source together in a culture medium under hypoxic conditions, wherein the hypoxic conditions comprise incubation in a carbon dioxide incubator at 37° C. with 1% oxygen concentration, and with a remaining concentration containing carbon dioxide and nitrogen,   wherein the MSCs from placenta are isolated and enriched by a process comprising of:
 collecting a chorion from the placenta; 
 mincing the chorion to obtain a minced chorion; 
 centrifuging the minced chorion to obtain a first pellet; 
 haemolysing the first pellet with a red blood cell lysis buffer; 
 centrifuging a mixture comprising the first pellet and the red blood cell lysis buffer to obtain a second pellet; 
 incubating the second pellet with a trypsin-ethylenediaminetetraacetic acid (EDTA) solution to obtain a trypsin-EDTA solution treated pellet; 
 re-suspending the trypsin-EDTA solution treated pellet in a minimum essential medium (MEM) or a fetal bovine serum to obtain a re-suspended mixture; 
 centrifuging the re-suspended mixture to obtain a third pellet; and 
 culturing, in a first culture vessel, the third pellet comprising mesenchymal stem cells in a minimum essential medium (MEM) supplemented with fetal calf serum, L-ascorbate-2-phosphate, L-Glutamine, an antibiotic, fungizone, and tylosin, 
   wherein the MSCs from an umbilical cord are isolated and enriched by a process comprising of:
 subjecting a processed umbilical cord to an enzymatic digestion with collagenase and hyaluronidase to form a mixture; 
 incubating the mixture containing the processed umbilical cord, collagenase, and hyaluronidase with gentle agitation; 
 adding trypsin to the agitated mixture and then incubating to obtain a digested suspension; 
 diluting the digested suspension with phosphate buffer solution (PBS) to obtain a diluted suspension; 
 filtering the diluted suspension to obtain a single cell suspension; 
 centrifuging the single cell suspension to obtain a pellet; 
 re-suspending the pellet in a washing solution comprising albumin, hydroxyethyl starch (HES), and citrate-phosphate-dextrose-adenine anticoagulant; 
 isolating cells containing MSCs from the washing solution; 
 seeding isolated MSCs with DMEM into a second culture vessel; and 
 incubating the seeded isolated MSCs until the seeded isolated MSCs reach 70-80% confluence. 
   
     
     
         35 . The method of  claim 34 , further comprising wherein the foetal polymix composition is formulated into an injectable composition. 
     
     
         36 . The method of  claim 34 , further comprising wherein the foetal polymix composition is formulated to be suitable for administering via an intravenous route, an intrathecal route, an intra-articular route, an intramuscular route, a derma roller, a mesogun, a local route, a periorbital route, an intravitreal route, or a retrobular route. 
     
     
         37 . The method of  claim 34 , further comprising wherein the foetal polymix composition is formulated to be suitable for treating disorders and diseases.