After the final isolation and re-suspension, the cells can be cultured and, if desired, analyzed to determine the number and viability in order to evaluate the yield. If desired, cells can be cultured without differentiation using эдьюкейшн a standard culture medium (eg DMEM, usually supplemented with 5-15% (eg 10%) serum (eg amniotic calf serum, horse serum, etc.). Methods of measuring viability and yield are known in the art (for example trypan blue exception).

In another aspect, the present invention relates to a lipid-derived lattice essentially devoid of cells, which includes an extracellular matrix material of adipose tissue. The lattice can be used as a substrate to facilitate cell growth and differentiation, both in vivo and in vitro, in embryonic or even mature tissues or structures.

After culturing the cells in a medium inducing differentiation for a suitable time (for example, from a few days to a week or more), the cells can be analyzed to determine if they really differentiated by acquiring the physical qualities of a given type of cell. One measurement of per se differentiation is telomere length, undifferentiated stem cells have longer telomeres than differentiated эдьюкейшн cells, so cells can be analyzed for telomerase activity. Alternatively, RNA or proteins can be isolated from the cells and analyzed (using Northern hybridization, from PCR, Western blot analysis, etc.) for the presence of markers characteristic of the desired phenotype. Of course, cells can be analyzed immunohistochemically or with dye using tissue-specific dyes.

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The material may include mature tissues or even whole organs, including types of tissues into which cells of the invention can differentiate (as indicated in this description). Typically, such material includes adipose, cartilage, cardiac, skin connective tissue, hematopoietic, muscular, renal, bone, pleural, visceral tissues, vascular tissues and the like. Of course, since cells can divide and differentiate to produce such structures, they can also form the germ of such structures. In the early stages, such primordia can be kept frozen in liquid nitrogen to further generate the desired mature structure or organ.

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The method includes maintaining the desired cell in an air-conditioned environment under conditions in which the cell remains viable. A cell can be maintained under any suitable culture conditions, such as are known in the art. Preferably, the method enables successive cycles of mitotic cell division to form a multiplied population.

Accordingly, there remains a need for a lattice material suitable for use as a substrate for the cultivation and growth of cell populations. Although a person skilled in the https://deveducation.com/ art is fully capable of practicing the present invention after reading the foregoing detailed description, the following examples will help to clarify some of its features.

Typically, such methods include transferring the cells to the desired tissue either in vitro (for example, as a graft before implantation or engraftment) or in vivo directly to the animal tissue. Cells can be transferred to the desired tissue in any suitable manner, which will usually vary according to the type of tissue. For example, cells can be transferred to a graft by holding the graft (or infusing it) together with a culture medium containing cells.

The exact conditions (e.g. temperature, CO 2 levels, mixing, the presence of antibiotics, etc.) will depend on other components of the medium and on the type of cell. However, optimization of such parameters is available to the ordinary person skilled in the art. In some embodiments, it is desirable that the medium does not contain the lipid-derived cells used to condition the medium, as described herein. However, in other embodiments, it is desirable that the lipo-derived cells are maintained in a conditioned medium and co-cultured with other cells of interest. Indeed, since the lipo-derived cells of the invention can express surface cell mediators of intercellular communication, it is often desirable for the cells of the invention and other target cells to co-cultivate under conditions in which two types of cells are in contact.

• On these lines, cells can be induced to differentiate into any of the aforementioned mesodermal lines of differentiation by co-culturing them with mature cells of the corresponding type or their predecessors.
• For chondrogenic differentiation, cells are preferably cultured at high density (for example, about several million cells / ml or using a micromass culture method) and also in the presence of low amounts of serum (for example, from about 1% to about 5%).
• Myogenic differentiation can be induced by exposing the cells to 10 μM to about 100 μM hydrocortisone, preferably in a serum-rich medium (for example, containing from about 10% to about 20% serum (bovine, horse, or a mixture thereof)).
• Chondrogenic differentiation can be induced by exposing cells to about 1 μM to about 10 μM insulin and from about 1 μM to about 10 μM transferrin, from about 1 ng / ml to 10 ng / ml transforming growth factor (TGF) β1, and from about 10 nM to about 50 nM ascorbate-2-phosphate (50 nM).
• For example, cells of the invention or population may be co-cultured with cells isolated from fetuses or embryos or in the presence of amniotic fluid.

A suitable lattice material is a secreted intercellular matrix material isolated from tumor cell lines (for example, Engelbreth-Holm-Swarm tumor matrix intercellular https://itstep.org/ tissue substance (matrix)). At the same time, other artificial lattices have been developed that may be toxic either to cells or to patients when used in vivo.

In one aspect, the present invention relates to lipoprotein stem cells substantially free of adipocytes and red blood cells, and clonal connective tissue stem cell populations. Cells can be used alone or in biocompatible compositions to generate эдьюкейшн differentiated tissues and structures both in vivo and in vitro. Additionally, cells can be expanded and cultured to produce hormones and to provide an conditioned culture medium to support the growth and reproduction of other cell populations.

Such an implant may be inserted or engrafted into the host to stimulate the generation or regeneration of mature bone within the patient. Such implants can be used to stimulate the growth or regeneration of muscle, fat, cartilage, tendon, etc. inside patients. Since the stem cells of the invention have a developmental phenotype, they can be used in tissue engineering. In this regard, the invention provides a method for producing animal material, comprising maintaining the cells of the invention under conditions sufficient to propagate and differentiate to form the desired material.

As discussed above, the cells, populations, lattices, and compositions of the invention can be used in the engineering and regeneration of tissues. The exact nature of the implant will vary according to the application where it will be placed. The implant may be or include, as described, mature tissue, or it may include immature tissue or lattice. Thus, one type of implant may be a bone implant, including a population of cells of the invention that undergo (or are primed for) osteogenic differentiation, optionally being seeded inside an array of suitable size and length, as described above.

This can be achieved, for example, by plating cells in the form of a heterogeneous population of cells on a suitable substrate for cultivation. Alternatively, the lipo-derivative cells of the invention can initially be grown to fuse, which will serve as a substrate for the second target cells for cultivation in an air-conditioned environment. Preferably, the stem cells are substantially free of other types of cells (e.g. adipocytes, red blood cells, other stromal cells, etc.) and extracellular matrix material; more preferably, stem cells are completely free of such other types of cells and matrix substance. Typically, cells of the invention will be obtained from human adipose tissue in accordance with methods such as those described herein. The present invention provides for the production of stem cells and lattice from adipose tissue.

Thus, for example, to assess adipogenic differentiation, cells can be stained with fat-specific dyes (e.g. oil red O, safarin red, Sudan black, etc.) or probed to evaluate the presence of adipose-related factors (e.g. type IV collagen, PPAR -γ, adipsin, lipoprotein lipase, etc.). Other methods for assessing the development phenotype are known in the art and any of them is suitable. Cells can also be used to generate monoclonal antibodies, which can then be used to assess whether they bind preferentially to this type of cell. Antigenicity correlation may confirm that stem cells differentiated along this evolutionary pathway.

Alternatively, cells can be seeded at the desired location within the tissue to base the population. Cells can be transferred in vivo using devices such as catheters, trocars, cannulas, stents (on which cell culture can be done), https://deveducation.com/ etc. The conditioned medium can be used to support the growth and reproduction of the target cell types, and the invention provides a method for culturing cells (in particular stem cells) using the conditioned medium.

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Other advances in tissue engineering have shown that cells can be cultured in spatially defined cultures to produce three-dimensional structures. Spatial certainty is usually achieved by using various cell-free lattices or matrices to maintain and direct cell growth and differentiation.