Membrane as substrate for growing cells of retinal pigment epithelium (versions), application thereof and method of inoculating such cells

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions deal with a membrane, used as a substrate for growing cells of retinal pigment epithelium, its application for supporting cells and a method of inoculating the cells on such a membrane. The characterised membrane is non-biodegradable and porous, covered from at least one side with a glycoprotein-containing coating, with pores with a diameter approximately from 0.2 mcm to 0.5 mcm, with a density of membrane pores constituting approximately from 1×107 to 3×108 pores per 1 cm2, and hydraulic conductivity of the membrane higher than 50×10-10 m sec-1 Pa-1, and having the maximal thickness of 11 mm.

EFFECT: claimed inventions make it possible to obtain a transplant for treatment of age-related macular degeneration.

19 cl, 4 dwg, 6 ex, 3 tbl

 

The technical FIELD

The invention relates to a membrane for use as a substrate for growing cells. In addition, the invention relates to a method of growing cells, methods of making membranes, and the use of cells. In particular, cells may be used in the treatment of age-related macular degeneration.

PRIOR art

Age-related macular degeneration (AMD) is a disease found in the elderly, which results in the thinning, atrophy and hemorrhage in the macular area of the retina. This leads to loss of vision in the Central (macular) vision, in particular the inability to see small details, to read or recognize faces.

AMD is divided into dry (not neovascular) and wet (neovascular). Wet AMD involves the growth of new blood vessels in areas where, presumably, should not be. The dry form is more common than wet, with approximately 85-90% of patients with AMD are diagnosed with the dry form of AMD. The wet form of the disease usually leads to more serious vision loss.

Dry AMD is the early stage of the disease and may result from the aging and thinning of macular tissues, deposition of pigment in the macula or a combination of these two processes. Dry m is multistrategy diagnosed in cases when sediments or of the decay products of decaying tissue, initially in the area of the macula begin to accumulate yellow spots called drusen. When dry macular degeneration may also cause a gradual loss of Central vision.

Dry AMD may progress to wet AMD, which under the retina, the growth of new blood vessels and the transudation of blood and fluid. This leads to transudation of permanent damage to light-sensitive retinal cells, which die off and create blind spots in Central vision.

Treatment of AMD is currently limited, however, some treatments can halt the progression of the disease or improve vision. Treatment of macular degeneration depend on whether the disease is in its early stages or has a dry form or correspond to a more advanced stage, the wet form, which can lead to serious vision loss. One method of treatment is the transplantation of cells from healthy peripheral region of the eye of the patient wet AMD in the affected area. Although it is effective, but there is a limit in the size of the affected area, which can be processed, and, in addition, the operation is time-consuming and not suitable for most elderly patients vozrastalo would be desirable to develop improved methods of treatment of both forms of AMD.

The authors of the present invention investigated the methods of substitution and transplantation of epithelial cells of the retina, in particular, using to retrieve the required cells stem cells. The inventors have developed a membrane that can be grown these cells and which can be transplanted into the eye, together with cells. The membrane is particularly well suited for growing cells, retinal pigment epithelial (RPE) and their derivatives, however, can also be grown and cells of another type.

Cells were grown on membranes before. Williams et al. (Journal of Materials Science: Materials in Medicine 16 (2005) 1087-1092) used a polyurethane membrane for the culture of RPE cells, however, it was found that in order to make possible the accession of cells, the membrane surface usually needs to be processed. Other groups have used polyester membrane.

The INVENTION

In accordance with the invention proposed membrane for use as a substrate for growing cells, the membrane is essentially nonbiodegradable and porous and has a pore diameter of from about 0.2 μm to 0.5 μm. Preferably, the diameter of the pores ranged from 0.3 μm to 0.45 μm.

The membrane is nonbiodegradable, to ensure that it will continue to support the cells after transpl is ncacii eyes. The term essentially nonbiodegradable means that the membrane will not break down for at least 5 years after the introduction into the organism, more preferably, at least within 10 years, even more preferably at least 15 years.

The pores of such a diameter allow for diffusion of nutrients and proteins, at the same time preventing cell migration through the polymer.

The density of pores is preferably lies in the range from approximately 1×107up to 3×108then 1 cm, more preferably in the range from 5×107up to 1×108then, on 1 see This density allows to provide the required levels of permeability, and also makes possible the vascularization. In particular, the size and density of pores are of great importance to ensure the movement of nutrients from one side of the membrane to the other, as well as to tumor blood vessels through the membrane. This is especially important after implantation. The polymer body can perceive the vascularization of the rich choroidal vessels. This is shown by the example of a rich vascular bed outside of the eye (Cassell et al., 2002; Patrick et al., 1999; Saxena et al. 1999, Peter et al. 1998), however, can proceed only when sufficient porosity (Menger et al., 1990).

Preferably, the hydraulic conductivity of the membranes was higher than 50×10 -10m s-1PA-1. In particular, the hydraulic conductivity of the membrane is preferably approximately 33 ml/min/cm2. This corresponds 801,21×10-10m s-1PA-1that eight times the hydraulic conductivity of a young macular corpse of Bruch's membrane. Such excess conductivity is useful as an artificial membrane relies entirely on passive processes. Being able to meet the needs of the next higher cell in terms of diffusion of nutrients, the membrane at the same time should not be a hindrance to the transfer of liquid from the basal side of the RPE layer, otherwise RPE will break away from the polymer surface. This makes sense as the reduced hydraulic conductivity of Bruch's membrane in the elderly presumably causes the epithelial pigment in AMD (Bird & Marshall, 1986).

Preferably, the membrane can be sterilized without deterioration of its properties using gamma irradiation, ethylene oxide, by autoclave treatment or ultraviolet radiation.

Preferably, the membrane can be sealed by ultrasonic welding, high-frequency currents or by insert molding. This allows you to attach to the membrane of the other layers, for example, when aadinath to the membrane pharmaceutical or cover layers. For example, you can attach more rigid biodegradable layer such as a layer of PLGA (copolymer of lactic and glycolic acids), to give the membrane stiffness for ease of introduction. Or may be attached layers, including pharmacological or biological agents, or layers, supporting other cells.

The membrane preferably has a maximum thickness of approximately 11 μm. More preferably, the thickness of the membrane is from 9 μm to 11 μm. The thickness of the membrane is chosen taking into account the occurrence of diffusion of nutrients, vascularization, as well as the possibility of an easy introduction membrane in the eye.

Thus, the proposed membrane for use as a substrate for growing cells, the membrane is essentially nonbiodegradable and porous and has a maximum thickness of approximately 11 μm. The membrane preferably is essentially flat, while its smallest size is preferably less than about 11 microns. It may vary in thickness in this dimension, but preferably has a thickness in the range of from 9 μm to 11 μm.

The membrane preferably has a maximum weight of approximately 1.5 mg/cm2. More preferably, the mass of the membrane ranged from 1.0 mg/cm2up to 1.4 mg/cm2. Minimum PR the Affairs of the tensile strength of the membrane is preferably 100 bar to provide adequate strength during surgery. The maximum tensile stress is preferably 300 bar, again to allow easy manipulation during surgery. The tensile strength of the membrane is preferably at least 10 psi.

Preferably, the membrane was hydrophilic. This gives the membrane a good wetting ability and allows you to easily attach cells and other required coverage.

The membrane preferably has a pH from 4 to 8, it is physiologically acceptable pH value.

The membrane preferably includes a coating on at least one of the parties. The coating preferably is a protein or glycoprotein, such as laminin, Matrigel, collagen, fibronectin and PLGA (copolymer of lactic and polyglycolic acids). The coating may also include pharmacological or biological agent that is attached to a component of the coating. For example, the coating can include a neurotrophic agent, an anti-inflammatory agent or an antiangiogenic agent.

In particular, the coating preferably contains laminin, in particular, laminin-1 or a fragment, such as IgVAV. In particular, the coating preferably contains more than laminin-1 than other protein or glycoprotein. Preferably, the coating has been included at the ore 30%, more preferably, at least 40% laminin, especially laminin-1. The coating is preferably applied with getting on the membrane concentration of laminin-1 approximately 40-45 m kg/cm2.

Accordingly, the proposed membrane for use as a substrate for growing cells, and the membrane includes essentially nonbiodegradable and a porous supporting layer, coated at least on one side a coating containing laminin-1.

The membrane is preferably made from a hydrophilic polymer. Can also be used and hydrophobic polymers made hydrophilic by irradiation of such a polymer by ultraviolet light. Particularly preferred polymers include polyesters such as polyethylene terephthalate, polybutylene terephthalate; polyurethanes and paleoceneeocene, in particular those which contain polycarbonate and polysiloxane, as well as those that are based esters and ethers; polyamides such as nylon; polyetherether, such as Sympatex; polycarbonates, such as Makrolon; polyacrylates, such as Perspex; poly(tetraphthalate) (PTFE); polysiloxane; polyolefin, such as polyethylene and polypropylene; and Polyoxymethylene (RUM), commonly known under the trademark DeIrin company DuPont. Particularly preferably, the membrane was manufactured is established with representation from polyethylene terephthalate or polybutylene terephthalate. According to another preferred variant implementation, the membrane is made from polyester.

The membrane used for growing layer of cells. The membrane preferably includes a layer of cells on the membrane. Cells can be any cells selected in accordance with the intended use of membranes and cells. Types of cells are any cells that can be grown in the form of a monolayer, and include retina cells, skin cells and endothelium cells, and induced pluripotent stem cells. Cells can occur from a variety of sources, for example the cells may be autologous cells taken from the body for transplantation back to this body, or may be cells, grown specifically for a particular purpose. Cells may originate from stem cells, in particular stem cells from human embryos. If the cells come from embryonic stem cells, preferably cells were available from embryonale source, such as a Bank of cells. According to a preferred variant implementation of stem cells from human embryos are pluripotent embryonic stem cells. More preferably, the stem cells of a human embryo to solenoidality were obtained by means other than killing a human embryo, such as the method of isolation of hESC (stem cells from human embryo), unveiled in the work Lonza et al., Nature, 444:481-485, Nov. 2006. Cells can be termed"immortal"cells, such as cells ARP-19. In particular, this invention is used to treat degenerative diseases, especially retina. Accordingly, the cells can be cells of the retinal pigment epithelium (RPE cells) or related cells, such as cells, differentiated with the formation of RPE cells or resulting from the differentiation of RPE cells (retinal derivatives) or their precursors. Such cells may include a photoreceptor cells, horizontal cells, amacrine cells or ganglion cells of the retina. Can also be used and other highly differentiated cells.

The membrane and the layer of cells preferably have a length and width of at least 3 mm × 5 mm, it is Preferable that the membrane and the layer of cells was at least 4 mm × 6 mm

When applying the cells to the membrane, it is useful to seed the cells tightly in order to reduce the possibility of dedifferentiation regulation. Ideally, cells should be seeded with a density of at least 200,000 cells / cm2more preferably about 250,000 cells / cm or with a higher density, such as the 300,000 or 350,000 cells / cm2.

Thus, the proposed porous nonbiodegradable membrane for use as a substrate for the colony of cells, and the membrane has a layer of cells on at least one of the parties, and cells seeded with a density of at least 200,000 cells / cm2.

Also presents a method for seeding cells on a membrane, comprising the stage of sowing the cells on the membrane with a density of 200,000 cells / cm2or higher.

The membrane preferably is a membrane in accordance with the invention. The cells preferably are highly differentiated cells, such as RPE cells.

Through the invention presents the use of membranes in accordance with the invention as a substrate for the colony of cells.

Also proposed membrane in accordance with the invention for use in therapy. In particular, the application of membranes for the treatment of age-related macular degeneration, retinal ruptures, macular degeneration, choroideremia, optic nerve atrophy Leber's disease and Stargardt.

In addition, a method for culturing cells, comprising the stage of:

a) seeding of stem cells from human embryo feeders from mouse embryonic FIB is oblastof (MEF), inactivated by mitomycin C, with a sowing density of at least 1,2×104/cm2or feeders of human fibroblasts with a sowing density of at least 6×103/cm2; and

b) maintaining the cells in an environment that includes the following componentry or their equivalents: modified Dulbecco Wednesday Needle with high glucose (4.8 g/l) (Knockout DMEM, Invitrogen) with substitute serum 20% Knockout serum replacement (Invitrogen), 1% solution of essential amino acids, 1 mmol L-glutamine (Invitrogen), 4 ng/ml human bFGF (Invitrogen) and 0.1 mm β-mercaptoethanol (Sigma).

Cultured cells are preferably fed every two days. Ten days after passage mode recharge preferably change on a daily recharge using the environment without a major growth factor in human fibroblasts.

Growing cells are preferably grown in a nutrient medium for at least 30 days, more preferably at least 35 days.

Media for cultivation of preferably contains at least 15%, more preferably at least 18% or at least 20% KSR (substitute serum Knockout serum replacement). In addition, the medium preferably does not contain plasmanet, LIF (factor inhibiting leukemia) and/or bFGF.

Cells form pigmented lesions. Foci predpochtitel is but remove and place on the extracellular matrix for the provision of connection and extension of Moncloa.

Hereinafter the invention will be described in detail only as an example with reference to the accompanying graphics.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

Figure 1 shows a portion of a biopolymer with HESC-RPE in situ in the eye of the pig through 1 month after transplantation (A). In: Light micrograph And showing the photoreceptors with internal/external segments near HESC-RPE on the biopolymer. With One polymer, transplantirovannam in the pig eye, the photoreceptors degenerate. Dye: Kizilay purple. The scale interval: A, 1 mm; B, 100 μm. This figure demonstrates the biocompatibility of the biopolymer in the experiments in vivo, as well as the fact that the monolayer of RPE cells is necessary to maintain the viability of photoreceptors.

Figure 2 is a confocal picture HESC-RPE on the biopolymer in the eye of the pig, 1 month engraftment. HESC-RPE forms a homogeneous pigmented surface near the photoreceptors pigs (rhodopsin, FITC (fluorescein isothiocyanate)) and covers RPE pigs. A number of photoreceptors present in HESC-RPE cells (A). The drawing shows that the donor RPE cells expressing RPE65 (TRITC). This figure demonstrates that transplantirovannam HESC-RPE operates in the usual manner in the experiments in vivo.

Figure 3 shows dissociatively HESC-RPE, transplanted trechnical the major dystrophic RCS rats, after 5 weeks on cyclosporine. Grafted human cells marked in red with its own unique surface marker antibodies in human cells (HSM). Labeled processes of HESC-RPE-derived cells bypass the outer limiting membrane and inserted into the synaptic layers of the owner. The selected square area on Figa increased by Figv This shows that the transplanted cells were able to maintain a good level of visual function in transplantirovannam eye (compared to retransplantation eye of the same animal). Visual acuity was assessed using own optimalisations equipment and expressed in cycles per degree (C/d).

Figure 4 shows cell growth on different membranes.

DETAILED description of the INVENTION

Example 1

Preparation of membranes

Thin polyester film was subjected to collimated charged particles from a nuclear reactor. With the passage of such particles through the polyester material they leave traces with high photoconductivity. Next, the polymer traces of dissolved etching solution to obtain a cylindrical pores. Changing the temperature and concentration of the etching solution, and the time it, get pores exactly the specified size.

The resulting membrane, not only is em a thin, opaque, microporous polyester film with a smooth, flat surface containing pores of a given diameter and number.

Obtaining cells from human retinal pigment epithelium (hRPE) from stem cells human embryonic (hESC)

Cell culture

hESC maintained in culture flasks coated with 0.1% gelatin, and seeded feeders from mouse embryonic fibroblasts (MEF), inactivated by mitomycin C (sowing density of 1.2×104/cm2), or equivalent feeders from human fibroblasts. Cells were kept at basal HESC-environment consisting of the following components: modified Dulbecco eagle medium with high glucose (4.8 g/l) (Knockout DMEM, Invitrogen) with substitute serum 20% Knockout serum replacement (Invitrogen), 1% solution of essential amino acids, 1 mmol L-glutamine (Invitrogen), 4 ng/ml human bFGF (Invitrogen) and 0.1 mm β-mercaptoethanol (Sigma). Immediately after retrieval from the cryopreservation of HESC lines maintained within passages 74 replacing the medium every 2 days. Cells were regularly split (1:5) to maintain colonies of undifferentiated HESC. (The evaluation was performed by contrast to markers SSEA3, SSEA4, TRA-1-60 and TRA-181. Standard testing performed at the University of Sheffield, confirmed that HESC had a normal karyotype (Shef1 (46XY) Shef7 (46XX)).

Differentiation and expansion of HESC-RPE

It was found that hESC-hRPE reliably formed when hESC colonies become supercomplete on MEF (mouse embryonic fibroblasts). When the boundaries of individual hESC colonies are merged together (10) days after passage), the mode change environment change from once or twice a day to once a day, using the basal medium hESC, discussed in detail above (without bFGF). This component was removed from the environment by a registered communications between bFGF and repression specifications RPE. Pigmented lesions appear in supercomputing hESC cultures between 1-2 weeks after the introduction of daily feeding.

The resulting pigmented lesions were cut mechanically by using the tip of a glass pipette and microsurgical knives. This approach is applicable in practice only in cases where the formed pockets have a diameter of at least 1 mm During this procedure, tried to cut off the surrounding non-pigmented material to the premises pigmented lesion on 35 mm Petri dishes coated with Matrigel™ reduced growth factor (BD Biosciences, diluted 1:30) or laminin. Each Cup was placed in a total of 10 pigmented lesions, then RPE cells were left to grow on Matrigel in the next 35 days (i.e. 5 weeks in the basal HESC medium without bFGF). At the time of this phase, the medium was replaced every 2-3 days. This period sufficient to produce a monolayer of pigmented layers of cells ranging in size from 2-3 mm. using this method, layers of RPE (approximately 1 cm) were maintained in the laboratory in vitro for 4 months.

To retrieve the target cells can be used in other ways and methods of differentiation, known in the art, including methods of obtaining RPE cells from khES-1, khES-3 cell lines (Osakada et al., (2008) Nat Biotechnol, 26, 215-24), as well as methods of obtaining RPE of N1, H7 and H9 cells (Klimanskaya et. al., (2004) Cloning and stem cells, 6, 217-245).

Seeding of human ES cells on membranes and the method of successive cultivation

Collecting cell environment:

Around the pigmented cell clusters did cut using sterile surgical knife to separate them from non-pigmented cell populations within the flasks tissue culture. Then the clusters were extracted using microdontia or sterile glass pipette. In some cases it was done with simultaneous spiritualium for ease of preparation.

Then pigmented clusters were joined together with their cultural environment in the Eppendorf tube. After that was carried out by centrifugation speed 12000-13500 rpm for 3-5 minutes, providing a dense deposition of clusters, currency environment with dissociative rastvoromeshalkami solution is prepared as follows: 90% non-enzymatic cell dissociation set problems the solution of PBS without calcium or magnesium (Sigma-Aldrich) and 0.25% trypsin. The rest is buffered by phosphate, Dulbecco saline without calcium or magnesium. Cell clusters were incubated in dissociating solution for 5-15 minutes at 37°C.

Using a pipette, carefully homogenized cell clusters to dissolve the pigment clusters. They were re-centrifugation speed 12000-13500 rpm for 5 minutes. Dissociating solution was aspirated without disturbing the sediment. After this was added to the culture medium and the existing clot cells re-suspended by homogenization. At this stage determined the density of cells using a counting chamber and counted the density of sowing.

Preparation of membranes:

Membranes were sterilized using a biological safety Cabinet with UV lamp for 30 minutes on both sides. Then they were placed inside the Cup for cultivation and used the appropriate box for their heaviness. With this installation of the membrane is then covered with Matrigel 1:30 (BD Biosciences) at 37°C for 30 minutes (method a thick gel)or at 4°C over night (method a liquid gel). Furthermore, it was successfully used laminin when the surface concentration of 1-10 µg/cm2. The advantage of laminin is that human laminin commercial is Eski available allowing you to use the xeno-free (without alien components)that are essential to achieve clinical standards of cleanliness (Lei et al., 2007).

The membrane fragments transplanted into the eyes of the pigs and the eyes of rats. The results of transplantation are presented in figures 1 through 3. Figure 1 shows the biopolymer fragment with HESC-RPE in situ in the eye of the pig one month after transplantation. This demonstrates the biocompatibility of the polymer, and that the monolayer of RPE cells is essential to maintain the viability of photoreceptors.

Figure 2 shows a snapshot of the HESC-RPE on the biopolymer in the eye of a pig in a month after engraftment. This figure shows that transplantirovannam HESC-RPE operates in the usual manner in vivo.

Figure 3 shows dissociatively HESC-RPE, transplantirovannam three-week dystrophic RCS rats, 5 weeks on cyclosporine. This figure demonstrates that the transplanted cells were able to maintain a good level of visual function in transplantirovannam eye (compared to retransplantation eye of the same animal). Visual acuity was assessed using own optimalisations equipment and expressed in cycles per degree (c/d).

Example 2

Seeding membranes

To find the optimum sowing density, vysokodispersnyi avannah cells RPE cells were sown on the membrane at different densities. After cells were observed to examine these characteristics of RPE cells, as pigmentation and morphology-type "cobblestones". The results are presented in the following table:

The sowing density (cells/cm2)Pigmentation in assessing the naked eyeMicroscopic examination
No. pigmented cells per 40x field of view of microscopeRegular morphology (a regular hexagon best =0)
70000no16Cells are too scattered for settlements
212000bright>4000,023
270000dark (good)>4000,024
391000the dark (best)>5000,0175

Example 3

Growing cells on different membranes

A number of different membranes experience and in terms of their ability to support cell growth. The results are presented in the following table:

TC polystyrenePLGA (copolymer of lactic and glycolic acids)Teflon (PTFE)PolyurethanesPolyester Dacron
DignityExcellent well-expressed surface growthGood, flexible, porousStrong, flexible, porousStrong, flexible, porousStrong, flexible, porous enough excellent surface growth. The pore size small enough to exclude the migration of cells of donor immune cells and immune cells of the host. Excellent surgical properties despite the subtlety (see thickness)
ThicknessThin film does not meet the requirements700-900 microns90-150 microns60-200 microns5-12 microns
The pore sizeNo data17-50 mcm approx. 0.3 to 0.5 μs20-200 microns0.4 µm
The density of poresNo dataHighHighLow1×108on cm2
HydrophobicityNoNoStrongNoNo
Cell affinityHighHighVery lowWeak moderateHigh

Example 4

Growing RPE cells

Morphology was assessed for translucent polymers using the direct fixation of the image on an inverted microscope with phase contrast. The opaque morphology of the polymers was evaluated by means of immune staining for synaptic markers (usually ZO-1).

For testing cell growth:

To assess cell growth used a combination of svetlucavi microscopy, immunochemistry with confocal microscopy, e is ectroni microscopy and analysis of the viability of the cells.

Analysis of the viability of the cells (analysis based Alamar Blue):

Various polyurethane polymers associated with commercial inserts from tissue cultures, as in the case of PLGA. Briefly, the primary filter is cut and the residual material otsmatrivali with sandpaper. After careful propulsive with PBS investigated polymer was found on the insert with the help of home cyanoacrylate glue (Loctite, Henkel Corporation Avon, Ohio). To determine the suitability of polymers based on polyurethane performed an analysis of the viability of the cells using Alamar blue, measuring the density of viable cells as follows: the analysis was performed as follows: cells were seeded on different surfaces with low density, using for optimum bonding medium containing serum. To ensure sufficient bonding of the cells were incubated for 24 hours at 37°C. they were Then rinsed twice in serum-free medium to remove the non-aligned cells, and then incubated for at least 12-24 hours in serum-free medium for synchronizing cells. Further, within a reasonable time growing culture. For measurements before adding a certain volume of a 10% solution of Alamar Blue in PRF-HBSS culture once was rinsed in a. sirovina salt Hanks solution, not containing phenol red (PRF-HBSS). Cultures were incubated at 37°C for 45 minutes. The supernatant liquid blue color will change the color to red fluorescent proportional to the number of living cells. The supernatant was collected in a 96-well plate and analyzed in the tablet reader on the subject of fluorescence (excitation 530-560 nm, emission 590 nm).

The results of the analysis of viability on the basis of Alamar Blue for different materials are given in the table below. In addition, we compared the growth of cells on polyurethanes and polyester, the results are presented in figure 4. All polymers based on polyurethane showed reduced cell growth in comparison with the inventive polyester filter.

TC polystyrenePLGATeflon (PTFE)PolyurethanesIndustrial polyester culture frequencyPolyester
The growth of cellsSuperbSuperbPoorModerateSuperbSuperb
MorphologyPoorPoorNo dataGoodGoodGood
Electron microscopy--Scattered individual cellsDetached/twisted layers of cells. This is further evidence of poor attachment of cellsMerging attached cellsMerging attached cells
Connecting staining (contrast) (confocal microscopy)YesYesNo dataYes (only solid polymer)YesYes
Polarity (confocal microscopy)YesYesNo dataPartial? (only solid polymer)YesYes/td>

These results were confirmed methods of immunocytochemistry and confocal microscopy, which revealed the presence of scattered cells in most polymers based on polyurethane. For one of the polymers based on polyurethane (solid polymer) was obtained good cell growth and connective staining, however, the cell layers were easily peeled off from the surface of the polymer.

From the results of the analysis can be seen as shown in the table above that the polyester has considerable advantages in comparison with other possible membrane materials.

Methods using PLGA (copolymer of lactic and glycolic acids):

Membrane of PLGA were obtained as a gift from ..Lavik and R.Langer (Department of Chemical Engineering, MIT, Cambridge, Massachusetts). Originally PLGA material acquired as Resomer® 5031-1 (Boehringer-lngelheim, Ingelheim, Germany) and synthesized asymmetric membrane of PLGA as described by these authors (Lu et al., 2000a and 2000b; Lavik et al., 2001 and 2002). Data asymmetric profiles allow adherence of cells to the smooth upper surface, at the same time allowing the cells contact the basal environment through the lower porous side of the polymer.

Asymmetric PLGA membrane was set in 6.5 mm insert Corning Transwell® after removal of the original membrane. The installation was carried out with the help of the private cyanoacrylate glue (Loctite, Henkel Corporation Avon, Ohio). The resulting inserts were sterilized by irradiating each side under a UV lamp in a fume hood with laminar flow within 30-60 minutes. Cells 28 ARPE-19 were seeded on membranes with a density of 90,000 cells/cm2.

In another series of asymmetric PLGA membrane was installed on an empty insert (as described above). Insert, and polystyrene tablets with tissue cultures (control) were coated with laminin in the standard working concentration (Sigma-Aldrich, prepared from murine tumor Engelbreth-Holm-Swarm). Tablets and insert were seeded with P30 ARPE19 higher density 180,000 cells/cm2to reduce the time on the confluence of the monolayer. The cultures were maintained using RPE medium based on DMEM with high glucose content twice a week.

Example 5

Coverage

Methods of culturing HESC:

HESC lines Shef1 and Shef7 kept in flasks coated with 0.1% gelatin, and seeded feeders of inactivated mouse embryonic fibroblasts (MEF) (Draper et al., 2002). Cells were kept at basal HESC-environment consisting of the modified Dulbecco eagle medium with high glucose (DMEM, Invitrogen) with substitute serum 20% Knockout serum replacement (Invitrogen), 1% solution of essential amino acids, 1 mmol L-glutamine (Invitrogen), 4 ng/ml human bFGF (Invitrogen) and 0.1 mm (3-mercaptoethanol (Sigma). After extraction with cryoconserved the AI HESC lines maintained within passages 74 by changing the medium every 2 days. Cells were regularly split (1:4)to save the colony of undifferentiated HESC.

The following coating was applied in accordance with the standard Protocol of the manufacturer, unless otherwise indicated: Matrigel (1:30), laminin, collagen IV, human collagen I, Puramatrix®, plasmanet, poly-L-lysine and uncoated as a control. Briefly, each floor was thawed at 4°C and diluted in PBS or serum-free medium. Tablets/paste was coated at 4°C, at room temperature or at 37°C for each case in accordance with the manufacturer's instructions. Then the matrix solution was removed and tablets washed or dried in the air according to the standard method. HESC-RPE colonies (primary colonies) were cut from the flasks with culture and placed in the environment, after which they were sown on tablets/paste.

To assess the attachment of HESC colonies and their distribution used visual observation. Monitor newly planted HESC in the interval from 24 to 72 hours after sowing showed that only the Matrigel and laminin resulted in colonial adhesion HESC-RPE, i.e. colonies were well immobilized and subjected to separation even with careful rinsing medium. In one series of experiments with human personsurname placental laminine was achieved immobilization of HESC-RPE is alone, while the other failed. All other coatings (IV collagen, human collagen I poly-L-lysine, Puramatrix®, plasmanet) was unsuccessful in this regard.

Example 6

Culture of RPE Shef 1 HESC

Cell culture

Shef 1 HESC maintained in flasks KZT25, coated with 0.1% gelatin, and seeded feeders from human fibroblasts (the optimum planting density of 2.25×105cells on KZT25 (9×103/cm2)). Cells were kept at basal HESC-environment consisting of the following components: modified Dulbecco eagle medium with high glucose (4.8 g/l) (Knockout DMEM, Invitrogen) with substitute serum 20% Knockout serum replacement (Invitrogen), 1% solution of essential amino acids, 1 mmol L-glutamine (Invitrogen), 4 ng/ml human bFGF (Invitrogen) and 0.1 mm β - mercaptoethanol (Sigma). After extraction with cryopreservation Shef 1 HESC was subjected to replacement of the medium every 2 days and was regularly divided (1:4)to save the colony of undifferentiated HESC (assessed by staining for markers SSEA3, SSEA4, TRA-1-60 and TRA-181).

Differentiation and expansion of HESC-RPE

It was found that HESC-RPE reliably formed when the colony SHEF1 HESC become supercomplete on the feeders. When the boundaries of individual HESC colonies are merged together (approximately 10 days after passage), the mode change environment change from once or twice a day to once a day, use what I basal medium HESC, details discussed above (without bFGF). This component was removed from the environment by a registered communications between bFGF and repression specifications RPE. Pigmented lesions appear in supercomputing HESC cultures between 1-2 weeks after the introduction of daily feeding.

Cells were cultured for another 5 weeks, after which pigmented lesions were cut mechanically, using the tip of a glass pipette and microsurgical knives. This approach is applicable in practice only in cases when get lesions with a diameter of at least 1 mm, so it can be useful for cell culture for more than 5 weeks. During this procedure, tried to cut off the surrounding non-pigmented material to the premises pigmented lesion on 35 mm Petri dishes coated with laminin-1. In each Cup can be placed in a total of 10-20 pigmented lesions, then RPE cells leaves grow in 2 ml of medium in the next 35 days (i.e. 5 weeks in the basal HESC medium without bFGF). During this phase, the medium was replaced every 2-3 days. This period sufficient to produce a monolayer of pigmented layers of cells ranging in size from 2-3 mm.

This method differs from other published methods for the following: 1. Use higher standardized to ncentrate KSR (20% as opposed to 8-15%, modified Lanza group) 2. During the production of RPE cells from supercomputing HESC cultures do not use plasmanet, human LIF or bFGF.

Dissociation and suspension HESC-RPE

Pigmented HESC-RPE were collected from the flasks KZT25, containing colonies of HESC-RPE feeders (passage 0) or of the growing layers of HESC-RPE feeders (passage 1). The cells were removed by cutting them with a sterile micronora and move them using sterile microdontia. Colony/layers HESC then aspirated and suspended in a mixture of HES-environment-bFGF (without bFGF) until further use.

The desired number of clusters were placed together with their growing medium in a test tube and centrifuged at speeds from 2400 to 3600 rpm for 5 minutes to facilitate the removal of the supernatant medium. The centrifugation was repeated as necessary to enable replacement solutions as follows: cells were twice washed in PBS and then incubated at 37°C for 20 minutes in 90% non-enzymatic solution for the separation of cells in PBS (copolymer of butadiene and styrene) (Sigma #C5914) and 10% solution of 10x Trypsin in PBS (trypsin from the pancreas of pigs, Sigma; final concentration of trypsin 0,25%). After this incubation period the cells were carefully crushed to full suspension, that is, to the absence of visible cellular schopl the deposits. They are then centrifuged to remove the buffer solution for the separation of cells, and then cells resuspendable in HES-environment-bFGF and placed in the incubator at 37°C until further use.

Installation of polymer layers on the culture insert

13 mm polyester filter disks with pore size of 0.4 μm and density of pores approximately 1×108pores/cm2were added to Transwell inserts instead of the original filters, using silicone elastomer vinyl based biological purity (Kwik-Sil, World Precision Instruments Inc. Sarasota, Florida). The insert has a thickness from 5 to 12 μm. The polyester had a weight of 1.2 mg/cm2. Insert once were rinsed in PBS, air-dried and irradiated on each side under a UV lamp in a fume hood with laminar flow for 30 minutes for sterilization. Then sterilized paste was stored in a sterile fume hood with laminar flow.

Floor and seeding prepared culture inserts

Sterilized insert covered diluted 1:30 with laminin-1 for 30 minutes at 37°C. (Laminin has a concentration of 43 ág/cm2and was incubated at 37°C for 30 minutes.) Laminin aspirated immediately before seeding cells. The density of cells in suspension HESC-RPE was measured using counting chambers Nubauer® dilution Trifanova blue. Staining Trifanova blue used to confirm the viability of the cells, which in each case exceeded 93%. Cells were seeded at optimum density 200000-400000 cells/cm2. Seeded cells were left to adhere for at least 24 hours and usually 48 hours before the first replacement environment. Subsequently, the medium was changed three times a week.

1. Membrane as a substrate for growing cells of the retinal pigment epithelium, which is nonbiodegradable and porous, coated at least on one side a coating containing glycoprotein, with a pore diameter of from about 0.2 μm to 0.5 μm, and the density of the pores of the membrane is approximately 1×107up to 3×108then, on 1 cm2and hydraulic conductivity of the membrane exceeds 50×10-10m s-1PA-1.

2. The membrane according to claim 1, characterized in that it further comprises a layer of cells of the retinal pigment epithelium or their predecessors on the membrane.

3. The membrane according to claim 1, characterized in that it is sterilized without deterioration of its properties using gamma irradiation, ethylene oxide, autoclaving, or under the action of ultraviolet radiation.

4. The membrane according to claim 1, characterized in that it is sealed using ultrasonic welding, high-frequency currents or molding the insert.

5. The membrane according to claim 1, having a maximum thickness of approximately 11 μm.

6. The membrane according to claim 1, having a maximum weight of approximately 1.5 mg/cm2.

7. The membrane according to claim 1, characterized in that it is hydrophilic.

8. The membrane according to claim 1, having a pH of from 4 to 8.

9. The membrane according to claim 1, characterized in that the coating is a laminin, Matrigel or fibronectin.

10. The membrane according to claim 1, including pharmacological or biological agent that is attached to a component of the coating.

11. The membrane according to claim 1, wherein the cells are cells of the human retinal pigment epithelium.

12. The membrane according to claim 11, characterized in that the cells are termed cells.

13. The membrane according to claim 1, characterized in that it is made of a hydrophilic polymer selected from polyester, polyurethane, paleoceneeocene, polyamide, polyetherether, polycarbonate, polyacrylate, polysiloxane, polyolefin, and Polyoxymethylene.

14. The membrane according to claim 1, wherein the cells are autologous cells.

15. The membrane according to claim 1, characterized in that it is made from polyester.

16. The membrane according to claim 1 for use in the treatment of age-related macular degeneration.

17. The use of a membrane according to any one of claims 1 to 9 for maintaining a colony of cells.

18. Method of sowing cells pet is based pigment epithelium or their precursors on membrane according to claim 1, includes stage sowing the cells on the membrane with a density of 200,000 cells / cm2or higher.

19. Membrane as a substrate for growing cells of the retinal pigment epithelium, made of polyester, which is nonbiodegradable and porous, with a maximum thickness of approximately 11 μm, coated at least on one side a coating containing glycoprotein, and the pore diameter of the membrane is about 0.4 μm, the density of pores of the membrane is approximately 1×108on 1 cm2and hydraulic conductivity of the membrane exceeds 50×10-10m s-1PA-1optionally containing layer of cells of the retinal pigment epithelium or their precursors.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to biotechnology, cell technologies and tissue surgery. A method for preparing a smooth muscle cell culture consists in cutting a blood vessel fragment, grinding it to a piece size of no more than 2 mm in any dimension, and incubating the pieces in a culture flask having its bottom preliminarily scratched and containing a culture medium containing 10% embyo foetal serum for at least ten days, but no more than 24 days at 37°C in the CO2 incubator environment; the method differs by the fact that the above blood vessel fragment is an ascending thoracic aorta fragment cut during the coronary artery bypass surgery; before the incubation, the above pieces of the ascending thoracic aorta fragment are kept in the culture medium containing 0.1% collagenase for at least 30 minutes, but no more than 60 minutes at 37°C and then washed in the cell culture medium.

EFFECT: invention enables preparing the cells of the aortic tissue directly from the patient's vital tissues for transplantology applications.

3 dwg

FIELD: medicine.

SUBSTANCE: method involves photodynamic exposure on a Vero cell culture of HSV-1 and HSV-2 infected African green monkey's kidney, on samples containing HSV-1 and HSV-2 and on a virus progeny; if observing a decrease of virus titre from a reference by 2 and more of lg TCD50/0.1 ml in the cell culture, samples containing the viruses, and in the virus progeny, the anti-herpetic action is considered to be effective.

EFFECT: using declared method enables more accurate assessment in vitro of the efficacy of the photodynamic therapy to study the mechanisms of various PDT options for the purpose of the further prediction of the PDT efficacy in the herpes-infected patients.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented solutions relate to field of immunology. Claimed are: pharmaceutical, containing peptide, obtained from HIG2 or URLC10, capable of inducing cytotoxic T-lymphocytes (CTL) by formation of antigen-presenting complex with antigen HLA-A0206. Described are isolated antigen-presenting cell and CTL and methods for their induction. Antigen-presenting cell is induced by contact of cell, expressing antigen HLA-A0206, with peptide, obtained from HIG2 or URLC10. Cytotoxic lymphocyte is induced by contact of CD8-positive T-cell with antigen-presenting cell, presenting on its surface complex of antigen HLA-A0206 and peptide, obtained from HIG2 or URLC10. Characterised are method and means of immune anti-tumour response induction by introduction to patient of medication, which contains peptide, capable of inducing CTL.

EFFECT: claimed inventions can be used in treatment of cancer disease, characterised by higher expression of HIG2 or URLC10.

12 cl, 8 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. Particularly, the present invention presents a method for providing the higher expression of markers associated with pancreatic endocrine cell line with the use a TGF-beta receptor agonist, such as activin A, activin B, activin C, GDF-8, GDF-11 or GDF-15.

EFFECT: present invention presents methods for stimulating the differentiation of pluripotent stem cells.

7 cl, 4 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, namely to leukolectins, and can be used in medicine. What is prepared is the polypeptide leukolectin characterised by SEQ ID NO:1-8. The recombinant preparation is ensured by using a nucleic acid coding it and integrated into an expression vector which is used to transform a host cell. Testing absence-presence or determining an amount of the polypeptide leukolectin are ensured by using an antibody or an antigen-binding fragment of a variable region of the above antibody which is specifically bound to the polypeptide leukolectin. The polypeptide leukolectin or the nucleic acid coding it are used as ingredients of a pharmaceutical composition in therapy of pathological disorders of skin and mucous membranes.

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16 cl, 19 dwg, 3 tbl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention concerns a method for propagation and differentiation of pluripotent cells. The described method involves the stages as follows: culturing the pluripotent cells and processing the pluripotent cells with GSK-3B inhibitor, wherein the above inhibitor represents 3-[1-(2-hydroxyethyl)-1H-indol-3-yl]-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole-2,5-dione.

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10 cl, 15 dwg, 11 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering, tissue technologies and medicine. The method for producing the mesenchymal stem cells of pluripotent lines of the human stem cells involves producing the embryoid bodies of the human pluripotent stem cells, attaching the embryoid bodies to a Petri dish to induce spontaneous differentiation of the embryoid bodies into the mesenchymal stem cells, culturing and proliferating the mesenchymal stem cells with maintaining the identity of the mesenchymal stem cells, wherein the induction of spontaneous differentiation at the stage is performed by looping an autologous cytokine with no outer cytokine added; there are also produced the respective cells, using them, a kit and a culture method.

EFFECT: invention can be used for producing the mesenchymal stem cells in regenerative medicine and cell therapy.

15 cl, 11 dwg, 3 ex

FIELD: biotechnology.

SUBSTANCE: adhering fraction of myelocariocytes is cultured in the nutrient medium with the addition of the stimulator - the protein kinase inhibitor p38.

EFFECT: invention enables to increase production of G-CSF by cells of adhering fraction of myelocariocytes.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to molecular biology, biochemistry and medicine. What is presented is a composition for inducing stem cell migration in fatty tissue of adults which contains human mesenchymal stem cells from a fatty tissue of adults in an amount of 1x107 to 1x1010 as an active ingredient which express a chemokine or growth factor receptor on a cell surface, or a secretory product of the above stem cells contain the chemokine or growth factor receptor; wherein the secretory product of the stem cells of the fatty tissue of adults represents adiponectin; and wherein the human stem cells of the fatty tissue of adults are first treated with a mixture containing chemokine or growth factor.

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8 cl, 9 dwg, 8 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: combined graft (CG) represents a dermal matrix (DM) prepared of a donor layer with self-specific multipotent mesenchymal stromal cells (MMSC) colonised on the surface thereof to the number of not less than 750-1500 thousand per 1 cm2. The group of inventions also refers to a method for preparing the combined graft involving bone marrow sampling from the patient, preparing a MMSC culture, applying a MMSC suspension in the concentration of not less than 1 million/ml on the DM surface at 1 million MMSC per 5 cm2 of the DM.

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11 cl

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine. What is described is a biological material containing: a) a liquid carrier containing a viscous solution containing at least one natural and/or semisynthetic polysaccharide and having a dynamic viscosity measured at 20°C and at shear rate D=350 s-1, within the range of 100 to 250 centipoise and/or kinematic viscosity within the range of 99 to 248 centistokes (measured in the same environment); b) a autologous or heterologous mesenchymal cell culture and/or c) platelet rich blood product.

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11 cl, 5 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine and concerns methods for creation a tooth of a required size, and repair of the lost teeth in the oral cavity by transplantation of the created tooth into the dental loss. Particularly, the method for the tooth creation of a required length in one direction involves the stages: placing the first cell aggregate and the second cell aggregate in a tight contact inside a supporting carrier, wherein the first cell aggregate and the second cell aggregate respectively consists of mesenchymal or epithelial cells; culturing the first and second cell aggregate inside the supporting carrier; the tooth size is corrected by a contact length of the first cell aggregate and the second cell aggregate in the same pre-set direction. A method for determining the contact length of the first and second cell aggregate for the tooth preparation of the required size involves preparing a number of structural types containing structures of various contact length of the first and second cell aggregate in the same pre-set direction; culturing each of a number of structural types inside the supporting carrier; measuring the tooth length prepared at the previous stage in one direction; correlating the given length and contact length providing the basis for calculating the required contact length of the first cell aggregate and the second cell aggregate.

EFFECT: group of inventions enables creating the tooth of the required size that enables preparing a single tooth that can be used as it is in the form of a transplant.

23 cl, 1 tbl, 6 ex, 13 dwg

FIELD: medicine.

SUBSTANCE: presented are engineered multilayered vascular tubes, comprising at least one layer of differentiated adult fibroblasts, at least one layer of differentiated adult smooth muscle cells. Further, any layer comprises differentiated adult endothelial cells. The said tubes have the following features: a ratio of endothelial cells to smooth muscle cells makes approximately 1:99 to approximately 45:55; the tube is deformable; an internal diameter of the tube is approximately 6 mm or smaller; the length of the tube is up to approximately 30 cm; the thickness of the tube is substantially uniform along the tube section. It is also provided that the vascular tube is free of any pre-formed frame. What is also described is a method of forming the above tubes.

EFFECT: engineering the therapeutically acceptable alternative vascular tubes withstanding physiological pressure, for transplantation into a patient's body and for use in testing cardiovascular drugs and devices.

29 cl, 4 ex, 3 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to a medical prosthesis to be implanted into the human body, particularly to a biological nasal bridge implant used in nasal bridge reparative surgery. The nasal bridge implant is made according to the method which involves sampling, sterilisation and cutting to the required size of the animal material in the form of cattle or swine tendons; cell recovery from the animal material; shaping of the animal material for establishing the required form of the nasal bridge; cross-linking of the animal material; antigen recovery of the animal material, alkaline treatment and introduction of the active substances promoting adhesion of a growth factor and stem cells; packaging of the implant into the container with a sterilisation solution.

EFFECT: preparing the biological nasal bridge implant.

16 cl, 2 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed to extract stem cells, including whirling of heparinised bone marrow with hydroxyethyl starch at the ratio of source ingredients of 1:2 with speed of 700g for 15 min. in the closed system of three haematological containers connected to each other with tubes with subsequent removal of fat admixtures and plasma into the container No.1, transfer of the mononuclear fraction of bone marrow, a part of supernatant and erythrocytes adjoining the interface of two media into the container No.2. Sludged erythrocytes and bone fragments remain in the main container, whirling of the produced sample with the speed of 900g for 15 min. in the container No.2 to produce cell material for intravascular introduction, at the same time after the specified whirling a part of supernatant is removed into the container No.1 without unsealing of the system.

EFFECT: production of paracrine effect of bone marrow mononuclear cells and provision of safety.

1 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce epithelioid cells of buffalo cow light foetus by means of long-term no-reseeding cultivation having high sensitivity to the virus of infectious rhinotracheitis of cattle, parainfluenza-3, viral diarrhea-disease of mucous membranes and adenoviral infection.

EFFECT: possibility to use in diagnostics of virus infections.

4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science; it is used in transplantology, traumatology, surgery and oncology, and may be used to fill bone defects. What is described is a bioimplant which represents a donor bone deimmunised with chlorine-containing oxidants, a surface of which is covered with a multifunctional bioactive nanostructured coating (MBNC) of M-Ca-P-C-O-N or M-Ca-CON, wherein M is a metal specified in a group consisting of Si, Ti, Zr, Hf, Nb, Ta, and colonised by recipient's mesenchymal stem cells (MSC).

EFFECT: MBNC-coated bioimplant is in line with the anatomical and morphological characteristics of the replaced bone, provides cell adhesion, no transplant rejection, accelerated formation of connective tissue and callus.

8 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science, namely to reconstructive surgery it aims at the applications in transplantation, traumatology, surgery, and oncology. What is described is a method for producing bioengineered constructs for bone defect replacement, which is based on a bone anatomically compatible with the replaced one which is deimmunised in 5-10% solution prepared of a dry mixture of sodium chlorite, sodium perchlorate, sodium chloride in a ratio of 7:2:1 and distilled water; it is coated with heterogeneous implantable gel and colonised with multipotent mesenchymal stromal cells recovered from the recipient's bone marrow using immunomagnetic separation technique.

EFFECT: method provides sizeable bone defect replacement, high strength, fast fixation and repair of the construct in the implantation region, causes no reject phenomena.

1 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: what is described is a composition for fistula treatment in an individual, including stromal stem cells of fatty tissue where at least approximately 50 % of stromal stem cells of fatty tissue making a composition, express CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 markers and where the contents of the stromal stem cells of fatty tissue in the composition makes at least approximately 3×106 cells/ml.

EFFECT: invention extends the range of products for fistula treatment.

8 cl, 7 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemistry of high-molecular compounds, namely to preparing film and spongy chitosan and collagen materials effective in human skin cell culture and wound grafting. A method for preparing the composite chitosan and collagen resorbable matrixes for human skin cell culture involves preparation of solutions polysaccharide chitosan and protein collagen, mixing them in preset proportions and formation of film and spongy matrixes of mixed polymer solutions. For this purpose, chitosan and collagen solutions in the concentration 1.0-4.0 wt % in a general solvent (aqueous 2 % acetic acid) are prepared, mixed in preset proportions, and the film and spongy matrix materials are formed from the prepared solutions. An amount of collagen in polymer mixtures makes 2.5-10 wt % (of chitosan). Further, films and sponges are heated to temperatures within 50-100°C for 1.0-5.0 h in an atmospheric environment.

EFFECT: use of the declared method allows preparing the film and spongy resorbable composite materials of natural polymers effective in human skin cell culture.

2 cl, 6 ex, 2 tbl

FIELD: medicine, transplantology, traumatology, orthopedics.

SUBSTANCE: the present innovation deals with the purpose to obtain osseous artificial block of vertebral bodies and trabecular bones of limbs at surgical treatment of traumatic lesions, degenerative-dystrophic osseous diseases, tumors, osteomyelitis and tuberculosis under conditions of systemic or local insufficiency of reparative osteogenesis. For local restoring the function of reparative osteogenesis in case of its different disorders, increased strength and rate of developing osseous block of vertebral bodies and maximal keeping achieved correction of vertebral deformation one should isolate stromal stem cells to cultivate and mobilize them upon fixing matrix out of porous titanium nickelide at 200 mln. cells/cu. cm, detect the implant's volume based upon measurements of pre-operational roentgenograms in standard projections by taking into account planned volume of operative interference, detect the quantity of stromal cells being necessary for developing local depot, calculate the quantity of desired medullary punctate, for the channel to apply the implant with mobilized stem cells.

EFFECT: higher efficiency.

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