The overall goal of this procedure is to isolate mesangiogenic progenitor cells or MPC from the human bone marrow by selective culture conditions. We're testing both their mesogenic and angiogenic potentials. Mesangiogenic progenitor cells are being identified at low percentage with a mesen-ki-ma-stro-mo cell culture optimized for clinical application.

When FBS as be replaced by human serum. This technique is unique in fact it allows very consistent isolation of a large number of alli-pol-ified NPCs suitable for sprouting angiogenesis and a generation of synchronized MSC cultures. We discovered this when after trips in digestion of MCS cultures in a talgrum serum few fried egg shape cell remain attached but differentiated into MCs after FPS treatment.

To isolate human bone marrow mononuclear cells or MNC begin by diluting five to 10 milliliters of fresh bone marrow up to a total volume of 50 milliliters with DPBS and mix the cell solution by inversion. Next equally distribute 25 milliliters of cells into two new 50 milliliter conical tubes and add 25 milliliters of fresh DPBS to each tube for a second mix by inversion. Then rest the cells at room temperature to allow the mineral bone fragments and fat to separate from the solution.

After 10 minutes use a pastuer pipette to carefully remove the floating fat and filter the cell suspensions through individual 70 micron filters without disturbing the mineral bone fragment pellets. To isolate the MNC layer 20-25 milliliters of the diluted bone marrow on top of room temperature 1.007 grams per milliliter density gradient medium in each of two 50 milliliter tubes and separate the cells by centrifugation. Then use a sterile pastuer pipette to pool the whitish rings of cells at the enter faces in a new 50 milliliter tube.

Wash the isolated mononuclear cells with fresh culture medium for five minutes at 400 times G and room temperature. Then re-suspend the pellet in five to 10 milliliter's of fresh culture medium. To isolate the MPC equilibrate hydrophobic T75 flasks with 15 milliliters of fresh culture medium at 37 degrees celsius and five percent carbon dioxide.

After 30 minutes seed four to six times 10 to the seven of the human bone marrow mononuclear cells per flask for a 48 hour incubation. On the second day discard the medium and non adherent cells from the flasks and feed the cells with 15 milliliters of fresh culture medium. Return the cultures to the incubator for six to eight days.

At the end of the incubation wash the adherent cells with fresh DMEM. Then incubate the cells with two milliliters of animal free proteas detaching solution for five to 15 minutes at 37 degrees Celsius. When the cells have lifted from the bottom of the flask add 10 milliliters of fresh culture medium to stop the reaction and collect the cells by centrifugation re-suspending the pellet in one to two milliliters of fresh culture medium.

After counting the MPC plate two times 10 to the four per square centimeter of the cells into tissue culture treated T75 culture flasks and adhere the cells overnight in 15 milliliters of fresh culture medium in the cell culture incubator. The next morning replace the cultured medium with 200 micro liters per square centimeter of misancolmulstromul cell or MSC expansion medium and culture the cells for another seven to 10 days. When the cultures have reached confluence subculture at three to five times ten to the three cells per square centimeter until reaching a new confluence of passage two at MSC then perform proteas digestion to collect the cells as previously demonstrated.

Plate two times ten to the fourth cells per square centimeter in tissue culture treated six well plates and fresh expansion medium. After growing the cultures to confluence again, mark two wells as no differentiation and refresh the expansion medium. Mark two wells as osteo blasts and replace the medium with 200 micro liters per square centimeter of standard osteo genic medium specifically designed for MSC differentiation.

Then mark two wells as adipo sites and replace medium with 200 micro liters per square centimeter of standard adipo genic medium specifically designed for MSC differentiation. Return the cells to the cell culture incubator for two to three weeks until the cells have differentiated. Then proceed to florescent detection of extra cellular calcium deposits and intro cellular lipid droplets accumulation.

To produce 3D spheroids, seed a large number of 20 micro liter drops of freshly isolated MPC suspension on the inner surface of a petri dish lid. When all of the drops have been placed carefully place the lid onto a petri dish base containing DPBS to prevent hanging drop evaporation and place the plate in the cell culture incubator overnight. The next morning add 300 micro liter allo-quats of standard extra cellular matrix proteins to each well of a four degree Celsius 24 well culture plate and gel the matrix proteins at their 37 degrees celsius for 30 minutes.

Add 700 micro liters of standard vascular endo thelial growth factor rich endo thelial cell growth medium onto the gelled ECM proteins. When the MPC have aggregated into 3D spheroids carefully invert the petri dish lid and use a sterile pasteur pipette to gently harvest the spheroids. Finally seed the spheroids into the culture wells and return the plate into the incubator.

Morphilogically MPC are characterized by their distinctive fried egg shape with a thick core region surrounded by a flat thin periphery exhibiting many philipodia. Polar elongation of the outer cell boundary is also often observed. MPC culture in MSC expansion medium results in the rapid but partial differentiation into proliferating flat and multi branched cells with residual undifferentiated MPC's still detectable.

After a further passage cultures result in a mono layer of exponentially growing MSC like cells. In the process the MPC reorganize their F-Actin into stress fibers while the Nestin expression becomes confined to a few rare cells. Flows-itom-etry of freshly isolated MPC shows over 95%of cells expressing CD31 and CD45 but lacking mis-en-com-al associated antigens CD90 and CD 73.

These latest antigens are highly expressed only after a complete differentiation into MSC. Confirming their mis-en-com-al nature when fed with differentiating medium for at least two weeks these cells can readily be induced into osteo blasts as indicated by their calcium deposits or adiposite's as apparent by the lipid droplet accumulation. Confirming the MSC nature of the predifferentiated cell cultures.

MPC typing is further confirmed in a sprouting angio genesis assay with the cells exhibiting an ability to invade a myrian extra cellular matrix protein gel from 3D spheroids after 24 hours of vascular endothelial growth factor stimulus. After one week, the invading cells migrate over an even greater distance an invasion capacity that is lost in second passage MSCs after the mesangenic differentiation. Once mastered the MSC cultures can be established in one hour if this is tackled properly providing between one or two million MPCs less than a week.

When attempting this procedure it's important to evaluate the performance of human CR batches before they use them in culture. At the moment our MPC isolation protocol is the only for obtaining high reproducable near pure MC culture for further downstream application. After it's development this technique paved the way for researches in the field of cell based therapies to explore the possibility to apply MPC in skeletal tissue regeneration.

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