Method for biosensor production (variants)

FIELD: biotechnology, in particular biosensors.

SUBSTANCE: claimed method includes production of sensitive cells producing in exited state signals being detectable by peripheral device. In one embodiment cells cultivated in cell cultures prepared form animal receptor cells are used as sensitive cells. In another embodiment as sensitive cells receptor cells which are functionally analogous to animal receptor cells cultivated in cell cultures prepared form animal stem cells are used. As peripheral device electrical signal receiver is used, wherein said electrical signal is generated by cell in exited state. Claimed invention is useful both in investigations and in industry.

EFFECT: biosensors with increased sensitivity, accuracy and integrity.

14 cl, 1 dwg, 4 ex

 

The technical field to which the invention relates.

The present invention relates to the field of biosensoric and relates to methods for producing biosensors intended for methods of detecting various types of radiation, in particular electromagnetic, and various chemical and biological substances in the analyzed environments, and can be used both for research purposes and industrial production.

The level of technology

It is recognized that an important basis of technical improvements in the areas of natural, medical and technical Sciences is the miniaturization of sensor devices, as well as increase performance and increase their sensitivity to the determined parameter. However, for a long time, this task was difficult due to the lack of relatively inexpensive devices and methods of their operation, operating in a wide temperature range, including physiological.

There is a method to determine the toxicity of individual substances and wastewater (Test toxicity in Chlamydomonas // Unified research methods water quality. M: CMEA, 1983, s-208). As the test organism (biosensor) used pure culture of Chlamydomonas gelatinosa. The method is based on the study of the effect of toxic substances on reproduction of Chlamydomonas gelatinosa. For this g is tovat sample solutions of different concentrations of these toxic substances in the standard nutrient medium and two control flasks with standard nutrient solution without adding toxic substances. Then in the prepared study and control samples make the same volume of the initial suspension culture of Chlamydomonas gelatinosa. Vessels with the test and control samples are placed in the Cabinet with fluorescent lighting, incubated for 21 days, browsing bulb and counting the organisms in the counting chamber every second day. The results of calculations are compared with the previously created table data. For clarity of display of digital data construct different graphs. Are taken into account only intact organisms.

The main disadvantage of this method of assessing results of Toxicological studies is that each body reacts individually to the presence of toxic substances. Therefore, the experiments often depends not only on the concentration of the toxic substance and the conditions of experience, but also from the presence in the tested group of individuals with different sensitivity.

Also known biosensor system for the determination of 2,4-dinitrophenol and nitrite ions in aqueous media, containing four biosensors, transducers which are immersed in the flowing, interconnected measuring cell electrodes Clark, as bioreceptors first and third biosensors contain cells of Rhodococcus erythropolis HL PM-1 immobilized on the carrier, che the fourth biosensor - cells of bacteria Nitrobacter vulgaris DSM 10236 immobilized on the carrier, the second biosensor includes a reactor column type immobilized on the carrier by bacterial cells of Rhodococcus erythropolis HL PM-1 as bioreceptor, with a Clark electrode of the second biosensor is placed at the reactor outlet (RU 2207377, 12 Q 1/02, 2003.06.27).

In (Huang, et al. Trends in Anal. Chem. 14(4) 158(1995) describes a static electrochemical method and electrode for monitoring the biochemical state of individual cells. The method requires the manufacture and manual positioning reference and the working electrodes inside a living cell. The method was used for detection of insulin, nitric oxide and glucose inside and outside of individual cells. The method is complicated and limited to the mandatory use of intracellular reductive biochemical reactions.

The closest in technical essence and the achieved result to the present invention is a method for the production of biosensors, including the production of sensitive cells, the signals from which are registered external device for detecting excitation (US 6,377,721, G 01 N 21/00, 23.04.2002). In the known method described some methods of using cells deposited on the chip for detection in the analyzed environments of those or other chemicals. It is important that in this case, the capture signal, postupayushie what about from the sensitive cells, by using the detection of the fluorescent radiation. Cells before application to the chip undergo genetic engineering modifications which result in cells introduce new genes encoding fluorescent or causing changes in the optical characteristics of the environment proteins. Cells in this case can also be further modified so that the signal on the detection of whom described the device, ran the fluorescent activity of a gene or causing changes in the optical characteristics of the environment of the protein or the protein with subsequent emission of fluorescent radiation and/or by changing the optical characteristics of the environment. These events, in turn, are detected by the external photosensitive devices associated with each particular cell or cell in which each individual cell using a light guide fibers.

Although many known methods of obtaining biosensors provide the tools applied to the chip as single cells and cell populations, to monitor the cellular response to the measured signals, but none of the known methods for biosensors no means use obtained from cell cultures, including cultures of stem cells, eukaryote the definition of intact or genetically modified receptor cells as the primary detector signal.

Disclosure of inventions

Task to be solved by the present invention is directed, is the creation and development of options for how you want to receive biosensors with improved characteristics.

The solution of this task is possible obtain a technical result consists in increasing the sensitivity, accuracy and reliability of detection of the excitation, the expansion controlled excitations.

Technical data the results for one version of the present invention are achieved by the fact that in the method of production of biosensors, including the production of sensitive cells, the signals from which are registered external device for detecting excitation, as a sensitive cells using cells grown in cell cultures prepared from the receptor cells of animals, and as an external device using the receiver electrical signal generated by the sensor cell for detecting excitation.

Alternatively in the method of production of biosensors, including the production of sensitive cells, the signals from which are registered external device for detecting excitation, as a sensitive cells use receptor cells that are functionally similar to cocktail recipes. what major animal cells, grown in cell cultures of stem cells in animals, and as an external device using the receiver electrical signal generated by the sensor cell for detecting excitation.

A distinctive feature of the present invention, one option is that as a sensitive cells using cells grown in cell cultures prepared from the receptor cells of animals, and as an external device using the receiver electrical signal generated by the sensor cell for detecting excitation. The difference between other option is that as a sensitive cells use receptor cells, receptor functionally similar to animal cells grown in cell cultures of stem cells in animals. The detection of the excitation signal of the biosensor includes capture specific signal or other type of sensitive cells, which generate the potential difference of the electric field. The potential difference is amplified standard equipment. Biosensors can be used for scientific purposes, in environmental monitoring and in industry for the manufacture of highly sensitive detectors of various types of excitation, for example spectra electric is magnitnogo radiation, and to quickly determine the concentration of certain chemicals in the analyzed media. Obviously, depending on the range of frequencies of electromagnetic radiation, depending on the specific controlled chemical or biological substances, etc., carry out the selection of appropriate sensitive cells. When this sensitive cells obtained in accordance with the present invention and grown in cell cultures, capable of virtually unlimited reproduction.

To increase the sensitivity for detecting excitation sensitive cells subjected to genetic engineering modification, for example by introducing into them the exogenous nucleic acid.

After receiving sensitive cells in accordance with the present invention, at least one sensitive cell is applied to the cell medium, which is preferably performed based on silicone, preferably in the form of wells. The hole has a diameter of from 1 to 200 microns and a volume of 1 femtolitr to 5 nanoliters.

List of figures

The drawing shows the General diagram of a device employing the biosensor obtained using the present invention.

The implementation of the invention

A device that implements the method in accordance with the present invention, contains the biosensor 1, the content is of ASI sensitive cells 2, the signals from register external device for detecting excitation 3. Sensitive cells can be grown in cell cultures prepared from the receptor cells of the animals. As a sensitive cells 2 can be used as receptor cells, receptor functionally similar to animal cells grown in cell cultures of stem cells in animals. As an external device used by the receiver 4 of the electrical signal generated by the sensor cell 2 for detecting excitation 3. Sensitive cells 2 it is advisable to have in cells 5, which are located on the carrier 6. The receiver 4 may contain any known units, optimizing the transmission and processing of the electrical signal amplifier, a calibrator, a memory block, the block comparison, etc. the Output of receiver 4 is connected to the device 7 visual indication that, in principle, can be combined with the receiver 4.

The practical implementation of the present invention is illustrated by the following examples:

Example 1. The creation of a biosensor based on immortalizing cell culture infrared receptors of jamkoveho body of the snake to detect thermal radiation.

Some species of snakes have a special receptor body, and allowing the catch to the temperature difference with the accuracy of 0.003 degrees Celsius. This body, called jankovym (eng. pit organ), provides animal dimensional vision radiating the warmth of the objects that you want to snakes when stalking prey in the dark. Studies have shown that the sensitivity in this case is provided with a layer of special sensitive cells of the nervous tissue functions infrared receptors (Jones BS, Lynn WF, Stone MO (2001). Thermal modeling of snake infrared reception: evidence for limited detection range. J Theor Biol. Mar 21;209(2):201-211).

In accordance with the present invention based on such sensitive cells can be created biosensor, high-resolution measurements of thermal radiation. To do this, from the body of the representative alcohology snakes (for example, the halys Pallas lat.: Ukraine halys halys) preparative allocated anchovy body. Retrieved tissue is crushed and used to create a primary cell culture of neural tissue according to the conditions described in the works (Ren D, Miller JD. Primary cell culture of the suprachiasmatic nucleus. Brain Res Bull. 2003 Sep 30; 61(5):547-53; Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, DenBesten P, Robey PG, Shi S. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002 Aug;81(8):531-5), using standard media and sera for growing narodnih cultures (for example, the combination of DMEM media and OPTIMEM with the addition of embryonic calf serum and growth factor neurons NGF (Engl.: nerve growth factor). Cells primary cultures immortalized, i.e. cases which are capable of unlimited number of divisions.

To do this, they are subjected to genetic engineering modifications. Using, for example, electroporation, lipofection transfection or viral vector to the cells is introduced exogenous DNA, containing the gene for the catalytic subunit of telomerase human cytomegalovirus promoter under the control of the inducible TET operator, as well as gene-reporter (e.g., green fluorescent protein, eng.: green fluorescent protein, GFP) and standard tape resistance to the antibiotic neomycin. Following a widely applicable procedure for separation of cells with integrated into the genome of the above-described construction starts the activity of a gene for the catalytic subunit of telomerase, which ensures the immortality of the cell culture. In more detail this way the immortalization of cell lines described in (Egorov HER, Terekhov SM, Vishniakova KhS, Karachentsev DN, Kazimirchuk EV, Tsvetkova TG, Veiko NN, Smirnova TD, Makarenkov AS EI'darov MA, Meshcheriakova luA, Liapunova NA, Zelenin AV. Telomerization as a method of obtaining immortal human cells preserving normal properties. Ontogenez. 2003 May-Jun; 34(3):183-92).

By growing the culture in an alternating electric field to achieve differentiation of dividing cells in receptor cells with normal morphology (monitored microscopically according to (Hirosawa K. Electron microscopic observations on the pit organ of a crotaline snake Trimeresurus flavoviridis. Arch Histol Jpn. 1980 Feb; 43(1):65-77). These cells are transferred into the wells of the silicon carrier connected to the EXT is m receiver electrical signal, and grown in standard nutrient media (as described in the work (Fan YW, Cut FZ, Hou SP, Xu QY, Chen LN, Lee IS (2002). Culture of neural cells on silicon wafers with nano-scale surface topograph. J Neurosci Methods. Oct 15; 120(1):17-23). When the detection of the measured signal (thermal radiation) is logged own electrical activity obtained in the culture of the sensitive cells. This activity is captured and amplified by the external device. Before using the biosensor must be calibrated in a controlled volume (in this example, increasing the temperature from 10 to 40 degrees Celsius in increments of 0.1 degrees Celsius). In model experiments the sensor accurately detects the ambient temperature in the physiological temperature range, and zooms in and out of heated objects.

Example 2. Creating a culture derived stem cells light-sensitive receptors of the green monkey biosensor for detektirovaniya electromagnetic radiation in the visible part of the spectrum.

Structure of the eye, that is, the photosensitive body green monkey, well-studied anatomically. The layer of light-sensitive cells of the retina is allocated preparative then get a primary culture of such cells (similar to the method of example 1). Stem cells are obtained by successive pereselenia cells, initially polucen the x from a primary culture, within 6 months. Alternatively, the selection of stem cells can be carried out positively stained fractions by hybridization with antibodies to marker proteins: Oct-4, Nanog, MELK, Tuj-1, Nestin, GFAP, and others (described in detail in: Maye P, Becker S, Siemen H, Thorne J, Byrd N, Carpentino J, Grabel L. Hedgehog signaling is required for the differentiation of ES cells into neurectoderm. Dev Biol. 2004 Jan 1; 265(1):276-90; Schwartz PH, Bryant PJ, Fuja TJ, Su H, O Dowd DK, Klassen, H. Isolation and characterization of neural progenitor cells from post-mortem human cortex. J Neurosci Res. 2003 Dec 15; 74(6):838-51). Selected stem cells grown in cultures analogously to example 1, in the selective conditions that cause the differentiation of stem cells into photoreceptor cells (cultured in an alternating electric field in the presence of standard factors of differentiation). The morphology of the obtained cells when examined microscopically. Prepared sensitive cells are then placed on a carrier, connected to an external device (analogously to example 1). Before the work of the technical part of the biosensor also need to calibrate, varying in the dark volume capacity of the incoming light radiation and its spectral characteristics.

Example 3. Creation on the basis of cultures of stem cells in the taste buds of rat biosensor for the detection of chemical substances in defined liquid medium.

From the language rats preparative allocated primary culture of the notches, enriched with cells of the olfactory receptors. Line stem cells are distinguished from primary cultures similar to the method described in example 2. Stem cells, prodifferentiating in cells of taste receptor, defined morphologically by using the methods of light and electron microscopy (see, for example, Mandairon N, Jourdan F, Didier A. Deprivation of sensory inputs to the humans bulb up-regulates cell death and proliferation in the subventricular zone of adult mice. Neuroscience. 2003; 119(2):507-16). Analogously to example 1 and example 2 prepared sensitive cells are placed in cells with a nutrient medium consisting of medium connected to the external device. As in the previous examples, is logged own electrical activity in sensitive cells. Calibration of the biosensor is more complex than for the previous two examples, as used sensitive cells contain complex ensembles of receptor molecules, each of which is responsible for the reception of a special group of chemicals. Thus, the descendants of various stem cells may have a different repertoire of sensitivity. Therefore, you should not allow uncontrolled mixing of sensitive cells of different descendants of stem cells in a biosensor. Calibration is carried out by adding a liquid medium, in which is put a biosensor, an individual the substances or mixtures of chemical substances, determining "bitter", "sour", "salty", etc. spectra sensations, or substances that have sharp or irritating odor.

Example 4. Creation on the basis of modified by genetic engineering methods cultures of stem cells from the olfactory receptors dogs biosensor for the detection of chemical substances in defined environments.

At the present moment is characterized by a significant number of protein coding genes at the molecular level causing sensitivity to smells. In this case, as a rule, each gene is required for optimal reception is strictly specific set of chemical substances in the environment (Lewcock JW, Reed RR. Neuroscience. ORs rule the roost in the system in humans. Science. 2003 Dec 19; 302(5653):2078-9). In this regard, the present invention can be applied to the development of biosensors with increased relative to the source of the receptor cells of a sensory capacity.

In this example for this is the culture of stem cells of the olfactory receptors of the dog (as in example 3). Received stem cells are then subjected to genetic engineering modifications, as a result of which they acquire additional copies of the genes of the receptor proteins. To do this, use the construction described in example 1, characterized in that instead of the gene for the catalytic subunit of the enzyme telomerase genes are proteins olfactory cocktail recipes. is s. Analogously to example 3 achieve differentiation of the obtained modified stem cells into Mature cells of the olfactory receptors, while the cells track microscopically or by using the methods of immunohistochemistry according to (Menco BP. Qualitative and quantitative freeze-fracture studies on humans and nasal respiratory epithelial surfaces of frog, ox, rat, and dog. II. Cell apices, cilia and microvilli. Cell Tissue Res. 1980; 211(1):5-29; Dennis JC, Allgier JG, Desouza LS, Eward WC, Morrison. Immunohistochemistry of the canine vomeronasal organ. J Anat. 2003 Sep; 203(3):329-38).

The application of the prepared cells on the carrier and calibration of the biosensor is carried out analogously to example 3 using a wide range of chemicals that can be added to the liquid medium, a thin layer covering the sensitive cells of the biosensor, and in the closed volume of the gaseous environment as minimal as possible, which put sensitive cell biosensor. In the latter case, you should increase the exposure time is defined components for the passage of their diffusion through a thin layer covering the sensitive cells in a liquid medium.

Creating a new generation of touch devices based on the use of biological receptors and/or their parts for detection of incoming signals is an important task of biotechnology. Such sensors could combine compactness, high sensitivity and environmentalists is a mini security work in the standard temperature range and relative nedorogovizny. Biosensors could be used in scientific, industrial and household purposes, and also for detection of different types of electromagnetic radiation and the presence in the analyzed environments of those or other chemicals. The development of nanotechnology allows the creation of technical means, effectively receiving, amplifying, converting and transmitting signals from a single receptor cells. Being arranged in an ordered structure, such cells would be a sensor with sensitivity set by selection of used cell receptors. However, existing in this area approaches is limited by the use of receptors, taken directly from the body of the donor, without the possibility of artificial developments cells-receptors of the reproducing cell cultures. In the present invention is characterized by the original method of obtaining receptor cells to create biosensors using cultures of stem cell lines and genetically modified lines and/or immortalized cell lines.

Thus, the methods of the present invention can be easily implemented using well-known knowledge, technologies and equipment. Indeed, at the present time in the world of science has achieved great success in the selection and the long-term cultivation outside the organization the mA stem cells, which can develop in almost any specialized cells, including receptor cells of the nervous tissue, and ultimately to give required for transplantation of cells, tissues, and in the long term and whole bodies (Benninger F, Beck H, Wernig M, Tucker KL, Brustle O, Scheffler (2003). Functional integration of embryonic stem cell-derived neurons in hippocampal slice cultures. J Neurosci. Aug 6; 23(18):7075-7083; Arsenijevic Y (2003). Mammalian neural stem-cell renewal: nature versus nurture. Mol Neurobiol. Feb; 27(1):73-98). Currently, however, the issue of stem cells is considered mainly from the point of view of medical use. It is essential that with the advent of nanotechnology has made possible the creation of technical means, commensurate with the scale of the cells (Xie Y, Sproule T, Li Y, Powell H, Lannutti JJ, Kniss DA (2002). Nanoscale modifications of in attachment and growth of mammalian epithelial and mesenchymal cells in vitro. J Biomed Mater Res. 2002 Aug; 61(2):234-245). In addition, methods have been developed for cultivation of cell cultures, including nervous tissue, silicone nanochip (Fan YW, Cui FZ, Hou SP, Xu QY, Chen LN, Lee IS (2002). Culture of neural cells on silicon wafers with nano-scale surface topograph. J Neurosci Methods. Oct 15; 120(1):17-23).

The method according to the present invention is industrially applicable, as evidenced by the following examples.

Some species of snakes have a special receptor organ that allows them to understand the difference of temperature with accuracy of 0.003 degrees Celsius. However, this organ called jankovym (eng. pit organ), PR is the animal volumetric heat vision objects what should the snakes when stalking prey in the dark. Studies have shown that the sensitivity in this case is provided with a layer of special sensitive cells of the nervous tissue functions infrared receptors (Jones BS, Lynn WF, Stone MO (2001). Thermal modeling of snake infrared reception: evidence for limited detection range. J Theor Biol. Mar 21; 209(2):201-211).

In accordance with the present invention based on such sensitive cells created biosensor, high-resolution measurements of thermal radiation. It is important that the sensitivity of this biosensor order of magnitude higher than the sensitivity of existing thermosensorik devices: 0.01 to 0.03 degrees Celsius. As described in the present invention the model (see the Example) was established and calibrated sensor device, really possessing at least a 15-fold increased sensitivity compared to the standard used in the art thermosensory.

Different types thermosensorik cells snakes (as well as thermosensory cells of various species of snakes), generally have sensitive molecules with different absorption spectra in the infrared region. When spectra are narrow enough to uniquely detect a particular infrared. This principle is based the creation of biosensor color imaging. Grown on met the dick in accordance with the present invention (see Example 1) culture thermosensorik cells with different excitation spectra were connected to the reader that maps the signal from a particular cell type specific color. When detection of excitation received a color signal indicating the presence of thermal radiation of certain wavelengths and their share in the total flow. A device that implements this technique is the first color imager-based biosensors.

Thus, technology in accordance with the present invention open extremely wide possibilities to create highly accurate and sensitive equipment of new generation, combining the potential of modern biotechnology, nanotechnology and computer science. These capabilities are further extended in connection with the possibilities of genetic engineering modifications of stem cells, which can allow multiple enhancing the natural sensitivity of receptor cells, by introducing new genetic information into cells-precursors of sensory cells.

1. Method for the production of biosensors, including the production of sensitive cells, the signals from which are registered external device for detecting excitation, characterized in that as a sensitive cells using cells grown in cell cultures, the sentence is olenych of the receptor cells of animals, moreover, as the external device using the receiver electrical signal generated by the sensor cell for detecting excitation.

2. The method according to claim 1, characterized in that the sensitive cells are subjected to genetic engineering modification.

3. The method according to claim 2, wherein the genetic modification is carried out by the introduction of sensitive cells by exogenous nucleic acids.

4. The method according to any one of claims 1 to 3, characterized in that at least one sensitive cell causing the cell media.

5. The method according to claim 4, characterized in that the cell media perform based on silicone.

6. The method according to claim 4 or 5, characterized in that the cell carrier is performed in the form of holes.

7. The method according to claim 6, wherein the hole has a diameter of from 1 to 200 microns and a volume of 1 femtolitr to 5 nanoliters.

8. Method for the production of biosensors, including the production of sensitive cells, the signals from which are registered external device for detecting excitation, characterized in that as a sensitive cells use receptor cells, receptor functionally similar to animal cells grown in cell cultures of stem cells in animals, and as an external device using the receiver electrical signal, generated the CSOs sensitive cell for detecting excitation.

9. The method according to claim 8, characterized in that the sensitive cells are subjected to genetic engineering modification.

10. The method according to claim 9, wherein the genetic modification is carried out by the introduction of sensitive cells by exogenous nucleic acids.

11. The method according to any of PP-10, characterized in that at least one sensitive cell causing the cell media.

12. The method according to claim 11, wherein the cell carrier perform the silicone-based.

13. The method according to claim 11 or 12, characterized in that the cell carrier is performed in the form of holes.

14. The method according to item 13, wherein the hole has a diameter of from 1 to 200 μm, and the volume from 1 femtolitr to 5 nanoliters.



 

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SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

7 cl, 2 dwg, 4 ex

The invention relates to biotechnology and can be used to obtain Malinov human IL-4 with an activating T-cell activity and reduced activating endothelial cells activity

The invention relates to biotechnology, in particular to the creation of transgenic plants with insecticidal properties

The invention relates to the field of genetic engineering and can be used in the biomedical industry

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

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