Complex system for collection, processing and transplantation of cell subpopulations, including mature stem cells, for regenerative medicine

FIELD: medicine.

SUBSTANCE: system includes a kit of disposable elements for transportation of liquid, which are preliminarily connected or include aseptic connectors for creation of connections between them in aseptic way, or are adapted for being aseptically connected. Kit includes three sets of disposable sterile elements: set for collection, set for processing and set for transplantation, packed in blister packing on carrier, such as tray, which has one department, containing each connected with other set of said kit. System can be applied for obtaining platelet concentrate for separate application. Method of collection and processing of cell subpopulation includes collection of isolated cells in collection chamber, connected with isolation device, processing of cells in centrifugal operation chamber, which is the same chamber as the chamber for collection, or which is connected with collection chamber, and collection of processed cells in chamber for reinfusion, which also is the same chamber as the operation chamber, or which is connected with operation chamber, and delivery of processed cell subpopulations back to the patient.

EFFECT: application of invention makes it possible to ensure automatic processing in closed system in on-line mode.

17 cl, 9 dwg

 

The technical FIELD

The present invention relates to the collection, automated processing and transplantation of cell subpopulations detected in the bone marrow, peripheral blood, umbilical cord blood, or adipose tissue, with the aim of local re-injection of these cells for tissue repair. Cell subpopulations in the typical case are adult stem cells or platelets, but more generally includes any of a subpopulation of cells such as erythrocytes and leukocytes. Such procedures can be carried out in hospitals or health care facilities that do not have laboratories for processing cells, and they can be carried out by the technical staff, not specializing in this field. The invention also relates to a new type of optical sensor for monitoring of cell subpopulations passing through the transparent tube.

PRIOR art

Stem cells are defined as cells that have the ability to clone and heal itself and which differentiate into numerous cell lines. Although embryonic stem cells come from embryos of mammals at the blastocyst stage and have the capacity to produce of course any differentiated cell in the body, adult stem glue the Ki are part of tissue-specific cells of the newborn organism, among which they are placed for differentiation. Adult stem cells have practical advantages over embryonic stem cells. Unlike the latter, they are not associated with any ethical issues, and may be isolated from the patient. They are abundant and found in various tissues of the human body. As shown by recent research, the most accessible sources of Mature stem cells are bone marrow, peripheral blood, umbilical cord blood, and possibly fat. These cells are capable of supporting, producing and of course replace differentiated cells within their own specific tissues due to physiological turnover of cells or tissue damage as a result of injury. This ability, known as cellular plasticity, has led to the development of therapeutic applications aimed at the regeneration of defective tissues in order to restore the physiology and functionality of the affected organ. As is well known already for many decades, adult stem cells can give rise to hematopoietic cells, but as discovered in recent years, they may also give rise to blood vessels, muscles, bones, cartilage, skin, neurons, etc. Such cells known as mesenchymal stem glue is key. In addition, platelets, prepared as a concentrate of platelets, can be used to accelerate wound healing and, therefore, may play a role in regenerative medicine to facilitate the reconstruction of tissues such as bone, skin or other tissues.

Hematopoietic stem cells were mainly used for transplantation in patients subjected to chemotherapy, to restore their blood. Originally isolated from bone marrow, they were isolated recently from peripheral blood or umbilical cord blood, and these latter have the highest proliferative capacity. Cells for transplantation require special handling, such as the division of cells, sometimes with the subsequent processes of selection and/or breeding. So far, these manipulations were conducted in well-equipped laboratories, specializing in working with cells, highly qualified staff who are competent in cell biology and Hematology. Such manipulations require labor-intensive intensive laboratory preparations involving centrifugation, the tubes division in the density gradient, often conducted in an open system to the risk of infection by bacteria, etc.

New perspectives offered by stem cells in region R is generative medicine, are doubts in relation to the practical problems of manipulation of these cells in the surrounding conditions, which is unusual for these techniques. One of the main problems is the lack of clean rooms, making possible the safe handling of cells. As examples cardiology, orthopedics and neurology, all of which are experimenting with the use of therapies based on stem cells, but there are no proper conditions for the processing of the cells. Therefore, there is a need for simple systems that can handle adult stem cells or any cell subpopulations automatically in a closed system, and quickly, to ensure that the treatment system cells in the "online" (on-line) at the bedside of the patient.

The INVENTION

In the invention, the proposed system makes possible the separation, collection, processing and transplantation of cell subpopulations, aimed at restoring tissue in regenerative medicine. The system according to the invention, as defined in paragraph 1, includes a set of disposable sterile items contained in the package, and contained elements of pre-connected or include aseptic connectors, or adapted to establish connections between them aseptic manner DL the software is functionally closed system. Such a system can be offered on a medium such as a tray, which includes separate sets for the procedure. Individual sets can be pre-connected or can be equipped with aseptic connectors to create connections between them aseptic manner, or can be connected using a sterile connecting device, such as SCD (from the English. sterile connection device)manufactured by Terumo operating by means of welding.

In the invention proposed a simple system for automated processing/concentration cell subpopulations in a closed system, which can ensure the processing system cells online at the bedside of the patient. Forms of embodiment of the invention set forth in the dependent claims.

In one form of implementation of the collection container used for collection of cell subpopulations from the patient, can be designed in such a way that it could be used as a camera for separation. Similarly, a tank (container)used for collecting the separated cells may be constructed in such a way as to serve as a container for reinfused to deliver cells back into the patient. Separation of cells can be aimed at the collection of leucocytes films or may be the using the method of separation based on density gradient, with the subsequent washing of the cells using the system described in EP-B-912250 (Claude Fell) and PCT/IB99/020523 (Biosafe). Another way of processing cells is the use of beads coated with monoclonal antibodies as described in WO 03/009889 (CellGenix/Biosafe).

Combined use of optical detector that can measure the absorption and reflection caused by the cells flowing in the transparent tube allows you to collect more accurately a specific cell subpopulation, like platelets, to obtain a concentrate of platelets.

Such a concentrate of platelets can be obtained in a separate procedure or as a by-product during the procedure, aimed at cell subpopulation. The invention also involves the use of systems described for the preparation of platelet concentrate for separate use.

Thus, the invention proposed a fully integrated system for intervention at the bedside of the patient, which minimizes the risk of infection through the use of a closed system. It offers a high level of automation and does not depend on special qualifications in the field of processing cells. It is suitable for manipulation with any source of cells (such as adult stem cells, platelets), but especially for stem cells casinogaming, in autologous or allogeneic environment.

BRIEF DESCRIPTION of DRAWINGS

The invention will be further described by example with reference to the accompanying schematic graphic materials, in which:

Figure 1 presents a diagram illustrating the General structure of the set for processing bone marrow according to the invention.

Figure 2 presents the notations used in figure 3-7 for illustration of the various components depicted kits according to the invention.

On Figa and 3B show two forms of implementation of the collection set, one without the filter installed and one with a filter installation.

Figure 4 shows the set for processing, which may be linked to aseptic connector set for collection, as shown in Figa or Figb, or set for transplantation, as shown in Figa or 5B.

On Figa shows the individual elements of the set for transplantation, and Figb shows the combination of elements set for transplantation.

Figure 6 illustrates the various combinations that make up a complete system.

Figure 7 presents the scheme of the all-in-one kit for the treatment of bone marrow, which rotate the working chamber includes separating the syringe, which is also used for the collection and transplantation of cells.

On Figa, 7B and 7B shows the activities of the respective configuration components of the kit, presented on Fig.7, respectively, for the collection, processing and transplantation.

On Figa, 8B and 8C shows the principle of the detection of cells using optical linear sensor using absorbing and reflecting properties of the data cells.

On Figg shows a top view of the optical linear sensor with the location of the LEDs and receivers.

Figure 9 shows a typical output signals of the optical linear sensor provided on Fig.

INFORMATION CONFIRMING the POSSIBILITY of carrying out the INVENTION

The invention relates to an integrated system, making possible the collection of cell subpopulations, processing/concentration and reinfusion product enriched in specific cell subpopulation, with the purpose of recovery or regeneration of damaged or defective tissue.

This method will be described in autologous conditions, which means that cells isolated from the patient and again enter the same patient. Such autologous treatment is usually preferred because there is no immunological reactions, or adverse effects due to incompatibility between donor and recipient. However, this principle will remain the same in the allogeneic environment.

According to the latest information, stem cells, and more specifically in mesenchymal STV is gross cell found in the bone marrow, although studies suggest that mesenchymal stem cells also exist in umbilical cord blood, peripheral blood, or even in the adipose tissue. Although this principle may also be applied to these different sources of stem cells described in this invention, the method relates to the treatment of the bone marrow.

This method consists, first of all, from a selection of bone marrow from the pelvic region under local anesthesia. Bone perforined using the extractor bone marrow, such as Tyco. Bone marrow is sucked off by using one or multiple syringes that are pre-filled with a certain amount of anticoagulant is heparin or citrate/phosphate solution. In the typical case, the collected volume of 50 ml, but may be another value. Aspirated bone marrow is usually transferred to a polyvinyl chloride (PVC) package to collect, either filtered or not, and can be placed on the stirrer. PVC package to collect then connect using aseptic techniques, preferably with the system described in EP-B-912250 and PCT/IB99/02052, and then spend accordingly, the separation and concentration of stem cells. Can be used and other centrifugal working chamber, for example, where the rotation axis is not parallel to the axis of the cylindrical working chamber, or used is of flexible containers.

In the patent document EP-B-0912250 (C.FELL)described a system for processing and separation of biological fluids into components comprising the set of containers for placing the biological fluid to be split, and the split components and possibly one or more additional containers for the auxiliary solutions. Hollow centrifuge working camera rotates around the rotation axis by bringing the working chamber into contact with the rotational drive. The working chamber has an axial intake/release for biological fluid to be processed, and the processed components of biological fluids. The intake/release leads to the space separation of variable volume, where all processing biological fluid by centrifugation. The working chamber includes a generally cylindrical wall extending from the end wall of the working chamber. This is basically cylindrical wall defines therein a hollow working chamber, which is hollow open cylindrical space located coaxially with the axis of rotation, and axial intake/release within the specified end wall coaxial with the wall, which is mainly cylindrical, open cavity of the working chamber. The working chamber contains in the wall, which is mainly cylindrical, coaxial concentration in the initial element, such as a piston. The space separation of variable volume is defined in the upper portion of the working chamber with the help of basically cylindrical wall and coaxial rolling element enclosed within which is basically cylindrical wall of the working chamber, and the axial displacement of the rolling element changes the amount of space separation, while the movable element is made with the possibility of co-axial movement in the working chamber to the suction of a given quantity of biological fluid to be processed, in the space of separation through the intake before or during processing by centrifugation and for extrusion processed components of the biological fluid from the space split over the issue during or after processing by centrifugation. Means are provided for monitoring the location of the rolling element, through which is controlled by the quantity of absorbed fluids and squeezing the split components. The system further includes a distribution valve mechanism for establishing selective communication between the working chamber and the selected containers or to install a working chamber and containers out of touch.

According to PCT/IB99/02052, this system is designed to operate in the regime of separate and indivisible transfer is, which provides better opportunities for application systems, including new applications that previously were not provided, such as the separation of hematopoietic stem cells and in General laboratory processing. Thus, the system can be designed so that it worked as follows:

in the split mode of the liquid can be sucked into the working chamber when the chamber is rotating or stationary; liquid Tenuta in the camera, centrifuged and separated into components, and the separated components are removed when the camera is rotated or, optionally, when it comes to the last divided component when the camera is stationary; and

in move mode, the working camera sucks in the fluid and pushes the fluid through a stationary camera. Starting valve mechanism may act to provide for transfer of certain amounts (doses) of a liquid from one container to another through the working chamber by moving the moving element without centrifugation or separation of liquid components, and a means for monitoring the position of the moving element controls the number of movable undivided liquids.

In the new application according to the present invention, which preferably use a camera section for the Deposit in accordance with EP-B-912250 and PCT/IB99/02052, separation may be aimed at the collection of leucocytes film, which makes possible the selection of stem cells without defining any specific cell subpopulations. Original sources described above, is injected into the chamber for separation by lowering the piston. After loading the product into the chamber for separation (as determined by optical linear sensor placed near the entrance to the chamber for separation) cycle of sedimentation (deposition) usually within 5-10 minutes gives a layer of leucocytes film between the plasma and the layer of red blood cells. When selecting first isolated plasma by moving the piston upwards. Optical linear sensor, for example, the sensor described with reference to Fig determines the cells that belong to the leucocytes film, and adjusts various parameters (speed selection, the selected volume, speed centrifugation) to optimize the output of the cells depending on the desired amount of time and processing constraints. Cells leucocytes film allocate a designated bag or bottle (depending on the configuration set for processing). The remaining erythrocytes or isolated in a dedicated package, or leave the camera (to save processing time). Depending on the parameters optimized during phase highlighted the possible leucocytes film, consecutive cycles (cycle) sedimentation/separation described above can be repeated. This will allow you to optimize a selected amount of leucocytes film depending on the final application of the cellular product.

In order to choose a more specific cell subpopulation, the principle described above (filling/sedimentation/separation), can be used with the same type of optical linear sensor, which defines more specific cell subpopulation by reflection and absorption. During the allocation of plasma absorption and reflection of the fluid are very low. When cells begin to flow in the tube, and then the absorption and reflection resulting product increases. The reflection also depends on the type and size of cells. This dependence allows to select a specific cell subpopulation. In the end, when the cell concentration is high, the absorption is at a maximum level, and the reflection is no longer possible. This can be used to identify cell subpopulations that have different sizes, such as platelets, and create a concentrate of platelets.

A preferred method for carrying out selection of cell subpopulation consists in the use environments of the density gradient, which focuses more specifically on a particular glue is full-time subpopulation. This will increase the purity of the product by reducing contamination by red blood cells and other unwanted cell subpopulations. Wednesday density gradient is chosen according to the target subpopulation. For example, for the isolation of mononuclear subpopulations can use environment based on a Ficoll™. In this case, the environment of the density gradient is first injected into the chamber for separation. Then slowly injected bone marrow by lowering the piston, in the typical case, when a speed of 5 ml/min to cause the cells to the environment layer of the density gradient. Erythrocytes and granulocytes will tend to pass through the layer of medium density gradient, whereas mononuclear cells and platelets will remain on the top layer. When the entire volume of bone marrow will be introduced, as determined using an optical linear sensor (Fig) in the upper part of the apparatus, the piston stop and a start phase of sedimentation within, for example, 10-20 minutes Extra thinner may be automatically performed by the system after complete aspiration of the product using isotonic connected to the system. Speed centrifugation can be increased to reduce the specified time of sedimentation. Then start the collection by moving the piston upwards. The liquid supernatant contains only the plasma. Then follow the first CL the weave which is the reason that tube to drain becomes opaque, which is determined using an optical linear sensor. This initiates the collection of mononuclear cells, which include the target stem cells. After reaching the volume, predefined operator menu, or when leaving the tube again becomes transparent, the collection of cells is stopped and the remaining contents of the chamber for separating collected in the waste package as long as the camera is not completely emptied. At this stage the camera to separate the washed all the remaining erythrocytes with an isotonic solution. The collected cells are re-injected into the chamber for separation with subsequent or previous flushing solution such as saline with albumin (alternative, can also be used phosphate buffered or another solution). Cells and the rinse solution are mixed. The piston will be stopped after reaching a specified amount, or when the camera is completely filled. Then carry out a new phase of sedimentation, during which the package is to collect can be washed using a supernatant obtained during sedimentation, removal of trace quantities of medium density gradient. The supernatant (consisting of rinse solution and environment the gradient of the and density), then squeeze. The process is stopped when the first cell will appear in the output tube, or can be repeated for best washing. At the end of the cell is collected in the collection container, which can be specially designed to facilitate further use of the collected cells. If necessary, the camera washed. Such cells can be used for re-introduction into the target organ of the patient, or can be further processed for the purposes of breeding or breeding. For these purposes, the system can resuspending cells directly in the desired culture medium.

Finer division than the use environment of the density gradient, is incubation of bone marrow in an environment that contains micro beads coated with monoclonal antibodies. This method of separation is described in the publication WO 03/009889 (CellGenix/Biosafe). The procedure is as follows. The product containing the beads, associated with a specific antibody is mixed with a blood product containing the cells of interest. After some time of incubation the beads are attached to the surface of target cells, leading to changes in their density. The mixture is then poured into the chamber to separate and begin the separation density in suspension, as described in earlier patents. When sedime the services will be completed, the supernatant is removed from the camera in the waste package, and the red blood cells then also drop. Of interest, cells labeled with beads and, therefore, having the highest density, will be the last cells that leave the chamber. They can be collected in a suitable container and, if necessary, and then washed to remove the antibody solution.

In the case of direct re-introduction after the processing, the collected cells may be connected, using aseptic techniques, the device, allowing transplantation to the patient. For use in cardiology such a device may be a balloon catheter used in angiography, for local re-injection of these cells, for example, in connection with the treatment of acute myocardial infarction. In this case, re-injected quantity of concentrated stem cells equal to 10 ml, graduated in 3 ml of pumping up the tank at regular intervals, ensuring the dissemination of stem cells. This method is described Zeiher et al. in the scientific article (TOPCARE - Circulation October 2002).

In the case of collecting a concentrate of platelets collected platelets can be used alone or in combination with thrombin, can also be obtained from the plasma of the patient, with the formation of trombotsitnoy gel, which will obligatorio healing. This trombotsitnoy gel contains growth factors, which are mainly to stimulate tissue regeneration alone or in combination with stem cells.

The entire method can be carried out at the bedside of the patient, and so is the way online, as schematically illustrated in figure 1. This provides significant advantages in safety, on-time delivery and reaction, and does not require any special skills in handling cells.

Collection of different target cell subpopulations can be carried out during the same collection procedures, but in order to apply these cell subpopulations in different time intervals during the same operation.

In the invention, the proposed system or "special packaging", which already contains a separate disposable sterile kits for implementing respectively the collection, separation and transplantation. Such packaging can be presented in a "blister"with 3 compartments, and each contains a disposable kit or set: one set for the selection of the bone marrow, one set for separating bone marrow, preferably of the same kind as the system described in EP-B-912250 and PCT/IB99/020523, and one set for the re-injection of cells. Each set may have several options, one is the most University, the sebaceous, providing a kit for transplantation, because it depends on the target tissue to be treated (e.g., bone, muscle, vessel and so on). Separate configuration of the kit illustrated in figure 2-7.

Perhaps these kits can be pre-connected together, or two of the three can be pre-connected if, for example, it is desirable to use a fully closed system. In the absence of a pre-connection practical solution is to use a specially designed aseptic connectors, such as system Medlock proposed PALL (link ACD) and described in patents US 3650093, US 5868433, US 6536805 and US 6655655, to ensure that the connections are performed under aseptic conditions, thus supporting the criteria of a closed system. Another possibility would be to connect the kit using a sterile connecting device. Any of the above configurations - pre-connected, or connected using aseptic coupling devices, or sterile connecting device to provide a functionally closed system. Such functionally closed systems eliminate the need for clean rooms or systems with laminar flow, which is a very important advantage in operating the whether in the surgical Department, which usually are not equipped to meet these requirements.

Another improvement of the invention consists in a container for bone marrow aspiration, which will act as a camera for separation in the second stage, called the working chamber. Its design is similar to the chamber for separation, as described in PCT/IB99/020523, and it can be equipped with a special needle to pierce the pelvic bone. It is pre-filled with anticoagulant or can be filled with anticoagulant before collection. After perforation of the bone marrow is sucked off by moving down the piston working chamber, powered by manual or electric vacuum source. Then the working chamber is placed in a centrifuge for a given installation, and the set consisting of an ordered sequence of tubes and packages, connect with the camera. After this can be initiated separation according to the method described above, using, for example, centrifugation Protocol leucocytes film. Another improvement of the invention consists in collecting the separated cells into a special container that can be easily connected or adapted to the system for reinfused cells back into the patient. Such container may be a graduated syringe, equipped with a Y-connector, the ima is holding one end, connected to a kit for the separation, and the other end provided with a connector syringe with Luer tip cemented tip for subsequent connection to the catheter.

On Figa shown set for collection without a filter, and PIGB - filter. Kit fee includes all necessary bone marrow aspiration:

a. The insertion point will represent, for example, the extractor bone marrow (for example, TYCO type) and in the typical case involves a needle to puncture (puncture) or bone needle to puncture the bones or veins. It can be directly connected with the rest of the set through a check valve or another valve.

b. One or more syringes (1...n), as required for aspiration.

c. The filter can be inserted into the collection for the collection to filter the bone marrow after harvest, as illustrated in Figb.

d. The collected bone marrow (filtered or unfiltered) are then stored in the packet for transfer before processing.

e. Additional syringe can be used to add the diluted product in the bone marrow and/or for flushing the filter if necessary.

f. Can be used aseptic connector (Pall ACD or similar).

Set for collection may be, for example, as follows.

C11 is Set to collect without filtercwith needle for boneaprewar the positive connected to the set for processing. The number X of syringesb.

C12 Set to collect without filter with needle for boneanot connected with pre-set for processing. The number X of syringesb.

C21 Set for collection filtercwith needle for boneapre-connected to the set for processing. The number X of syringesb.

C22 Set for collection filtercwith needle for boneanot connected with pre-set for processing. The number X of syringesb.

In all these configurations, the needle to the bone may be replaced with a needle to puncture veins.

Figure 4 shows the set for processing, which is adapted for connection with the previously described set to collect through aseptic connection.

a. Aseptic connection can be made through aseptic connector (such as Pall ACD or other) or needle connector under aseptic conditions.

b. Optional drip chamber can be used to prevent bubbles penetrating into the processing pipeline.

c. The direction of the fluid can be selected using the panel shut-off valves, which in this example are three shut-off valve, or other type of valve, for example, a multiport valve.

d. For the method of separation used the camera to separate, for example, described in EP-B-912250 and PCT/IB99/02052.

e. Dopolnitelnye) product(s) (isotonic, cultural environment and others) is injected by means of a tube provided with one or more compounds (needle connectors, etc. - 1...n).

f. Package for related products or waste used for unnecessary product and enter the environment of the density gradient.

g. Output tube with a possible intermediate service sends the product to set for transplantation.

h. Aseptic connector (Pall ACD or similar) may be used on the pipeline.

The main options set for processing include:

P1 set for processing without intermediate package

P2 set to process with intermediate package.

On Figa shows a possible individual elements of the set for transplantation, and Figb shows one of the possible combinations of elements of the set for transplantation. Set for transplantation is adapted for connection with the previously described set to handle through the aseptic connection and will contain the final product for transplantation. Aseptic connection can be made through aseptic connector (such as Pall ACD or other) or needle connector under aseptic conditions.

Set for transplant may include:

T1 package

T2 vial for collecting

T3 syringe

T4 special device for transplantation (e.g., a catheter for myocardial MIC is RDA)

T5 combination of T1-T4.

Generally speaking, set for transplantation will include as a minimum, at least one special device for transplantation T14, which can be combined with different combinations of other components, for example, T1 package or vial to collect T12, and/or syringe T3.

Figure 6 illustrates various combinations to create a full system. A complete system may consist of, for example, from any combination of set collection (C11-C22), set for processing (P1 or P2) and transplantation (T1-T4)as described above.

Figure 7 presents the scheme of the all-in-one kit for the treatment of bone marrow, which rotate the working chamber (chamber for processing)b3for example, described in EP-B-912250 and PCT/IB99/020523, includes separating the syringe, which is also used for the collection and transplantation of cells. Set to collectandconsists of the insertion point, for example, extractor bone marrow (for example, type TYCO)with aseptic connector for connection of the working chamberb3. Set to processbincludes shut-off valveb1connected to the packageb2for washing and packageb4environment density gradient /waste, and also the camera b3 for selection/treatment/transplant, which is connected aseptic connector with shut-off valveb1or set the m collection andor set transplantsc. Set for transplantationwithconsists of a special device for transplantation (e.g., catheter for myocardial infarction)with aseptic connector for connection of the working chamberb3.

On Figa, 7B and 7B shows the current configuration of the components of the kit Fig.7, respectively, for the collection, processing and transplantation.

On Figa working chamber for collecting/processingb3connected aseptic connector with the insertion point system for collection ofandso that the working chamber serves to collect the selected stem cells. Induction of stem cells is controlled by displacement of the piston working chamberb3.

On Figb working chamber for collecting/processingb3connected to its aseptic connector with shut-off valveb2that selectively connects it with the package for flushingb2and with service to the environment of the density gradient/wasteb4for the above-described processing operations, culminating in the return of processed/concentrated stem cells into the working chamberb3. Therefore, the working chamberb3serves as a chamber for reinfused.

On FIGU shows the working chamberb3after disconnecting from the shutoff valveb1set for processing, the United aseptic connector with the device d is I transplant from a set for transplantation. In this configuration, reinfusion treated stem cells to the patient can be controlled by displacement of the piston working chamber for processing/reinfusedb3.

This form of exercise based on the use of aseptic connector for selective connection of the working chamberb3with a set to collectandor with the rest of the set for processing through a check valveb1or set transplantswithfor further processing of the collection, processing and reinfused. This provides a particularly compact system that does not include any unused items and easy to use.

On Figa, 8B and 8C shows the principle of the detection of cells using optical line sensor LS using absorbing and reflecting properties of the cell through the transparent tube. On Figa shows the configuration of the tubes containing a transparent fluid, where the light from the LS is not reflected and passes directly into a direct detector R-axis light. On Figb shows the configuration of the tubes containing the cells in suspension in a transparent liquid; in this case, the light is reflected by the cells in random directions and is captured as a direct detector R and lateral detector R angled at approximately 90° to the axis. On FIGU shows the configuration of the tubes containing naprosy the ing liquid, where the light is not reflected. On Figg shows a top view of the optical linear sensor with the location of the light emitting diode (LED) and the receivers, in particular, showing the position of Direct Blue (F), Side Blue (L), Direct red (F) and red Side (L) light.

Figure 9 shows the characteristic signals of the optical linear sensor, which are recorded from the "direct" and "lateral" sensors. The information obtained from the "side" of the reflected signals can be used as a starting signal to start or stop collecting. The output value of the sensor (Y-axis) represents the allocated amount of leucocytes film (sun, from the English "buffy-coat") in percent of the maximum level. The X-axis contains information about the volume passing through the tube (in percentage of the total volume).

It should be understood that this invention may be implemented in various forms without deviating from its essence or essential features. Scope of the invention defined in the accompanying claims, and not in the description preceding it. Therefore, assumes that all forms of exercise, which fall under the terms of the claims are covered by the claims.

1. The system for selection, collection, processing and transplantation of cell su the populations, including adult stem cells and platelets, in particular, for the recovery of bodies in regenerative medicine, including a set of disposable sterile items for transporting liquids containing
the device for selecting, for example, includes needle to puncture the bones or veins, for the isolation of bone marrow or other sources of cell subpopulations from the patient;
at least one camera for capturing, processing and reinfused cell subpopulations isolated from a patient, including a camera for collecting, pre-soedinennoi or connected to the device for selecting, for a collection of cells isolated from a patient using the device for selection; a working chamber adapted to combine with technological equipment to carry out the processing and transfer operations of the collected cells; a camera for reinfused to store the processed cells, which are to be delivered back to the patient; and chambers for collection, processing and reinfused are separate and pre-connected or connected to each other, or multi-purpose working the camera provides the combined functions of a camera for collecting and processing chambers for processing and reinfused or camera for collecting, processing and reinfused;
device for transplantation pre-connected or connect the accused with the camera for reinfused for delivery of the treated cells back into the patient, characterized in that the set of disposable sterile items contained in the package, and contained elements of pre-connected or include aseptic connectors or adapted to establish connections between them aseptic manner to ensure a functionally closed system.

2. The system according to claim 1, characterized in that the set of disposable items Packed in blister packing media, such as a tray, with the blister pack has one branch containing the whole set of compounds, or many departments, each of which accommodates a part of a set that includes aseptic connector for connection to another part of the set.

3. The system according to claim 1, characterized in that the set of disposable items includes three sets of disposable items: set collection, processing and transplantation.

4. The system according to claim 3, characterized in that the kit fee includes an extraction device for bone marrow, single or in combination with at least one syringe, package to migrate, forming a chamber for collection, and, optionally, aseptic connector for connection set for processing.

5. The system according to claim 4, characterized in that the kit for collecting further includes a filter that is attached or attachable between the syringe and what acetam to transfer.

6. The system according to claim 3, wherein the set of processing includes the working chamber and at least one disposable container, which is connected with the working chamber via at least one shut-off valve or a multiport valve providing selective transfer of fluid into the working chamber and the working chamber and single(s) container(s) and/or single-use(s) of the container(s), and the working chamber is connected with a set for collecting or has aseptic connector for connection set for collection, or adapted to create an aseptic connection while the working chamber is also connected to the set for transplantation or has aseptic connector for connection set for transplantation, or adapted to create aseptic connections.

7. The system according to claim 6, wherein the set of processing further includes a tube provided with one or more connectors for connecting additional containers with shut-off valve or a multiport valve.

8. The system according to claim 3, characterized in that the set for transplantation includes at least one device for transplantation, or a combination of at least one device for transplantation with at least one of the following: a package for the collection vial to collect and syringe.

9. The system according to claim 1, the tives such as those that the working chamber is a hollow centrifugal working chamber with the intake/release for the cells to be processed, and the processed cells, and specified the working chamber contains a mobile element, which defines the space separation of variable size to host cells, and the specified item is made with the possibility of moving to suction the selected number of cells to be processed in the camera to separate through the inlet and for extrusion of the treated cells from the separation cell through the release.

10. The system according to claim 9, characterized in that the centrifugal working chamber is generally cylindrical and is made with the possibility of rotation around the axis of the cylinder, and the movable element is a piston in direct contact with the liquid, movably mounted in the working chamber of the centrifugal.

11. The system according to claim 1, characterized in that it includes
device for separation of bone marrow or other sources of cell subpopulations from the patient, and the specified device is connected through aseptic connection of the working chamber to collect stem cells, selected with the help of this device in the working chamber;
at least one disposable container, which is connected with the working chamber via at least one of apory valve or a multiport valve, providing selective transfer of fluid into the working chamber and the working chamber, and single(s) container(s) and/or single-use(s) of the container(s), and the working chamber can be connected to shut-off or multiport valve through aseptic connector; and
at least one device for transplantation, connected with the working chamber by means of aseptic connection to the working chamber acted as a camera for reinfused processed for delivery of cells to the patient.

12. The system according to claim 1, characterized in that the working chamber is adapted to obtain a product enriched in specific cell subpopulation, including adult stem cells and platelets.

13. The system according to claim 1, characterized in that the working chamber is adapted for the separation of stem cells using the method based on the density gradient with subsequent washing of the cells.

14. The system according to claim 1, characterized in that it is adapted for processing of stem cells using beads coated with monoclonal antibodies.

15. The system according to claim 1, characterized in that it includes an optical linear sensor for registration of differential light reflection cell subpopulation passing through the transparent tube.

16. The application of the system according to any one of the claim 1-15 to obtain a concentrate of platelets for separate use.

17. The method of collection and processing of cell subpopulations isolated from man, for transplantation to humans, in particular, for the recovery of bodies in regenerative medicine, including the collection of selected cells in the chamber for collecting connected with the device for selection by which distinguish cells, particularly bone marrow containing stem cells; the cells are processed in a centrifugal working chamber, which is the same camera as the camera to collect, or which is connected with the chamber for collection; and the collection of treated cells in the chamber for reinfused, which is the same camera that the working chamber, or which is connected with the working chamber; a chamber for reinfused connected to the device for transplantation connected with camera for reinfused to deliver processed(s) cell(s) subpopulation(s) back to the patient, characterized in that it is carried out by using the system according to any one of claims 1 to 15.



 

Same patents:

FIELD: medicine.

SUBSTANCE: there is offered a method for producing adult stem cells with multipotent properties that involves recovering thereof from human brown adipose tissue and cultivating in a nutrient medium containing N-acetyl-L-cysteine, as well as a method for long-term maintaining these cells in the undifferentiated condition due to formation of spheres in a CORM-2-containing medium. The adult stem cells produced by the offered method are characterised by high proliferation rate, positive CD73, CD90, CD29, CD44 and CD 105 immune response, negative CD33, CD34, CD45, CD4, CD31, CD62p, CD14 and HLA-DR immune response and ability to differentiate in cells of mesodermal origin. Particularly, disclosed is a possibility of producing nervous cells, cartilaginous cells, osteogenous cells, adipose cells and insulin-producing pancreatic cells of said adult stem cells under the invention.

EFFECT: method improvement.

12 cl, 15 dwg, 2 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: solution of viable breast cells after enzymatic degradation in a collagenase solution of analysed tissue at temperature 37°C for 30-35 minutes, are applied on a biomaterial of a native form of hyaluronic acid, stored at room temperature and constant humidity 30-50 %, and cell viability is controlled by discoloration of cell monolayer.

EFFECT: invention allows producing a viable cell monolayer and providing their vital activity for 2-3 hours.

1 tbl, 2 dwg

FIELD: medicine.

SUBSTANCE: eyeball is enucleated in autopsy, washed in an alcoholic solution and in Ca2+ Mg2+ -free Hanks' solution with an antibiotic added; an anterior segment of an eyeball is removed along a dentate line, a vitreous body and a neutral retina are separated from a pigment epithelium; an eyecup is filled with Ca2+ Mg2+ -free Hanks' solution with EDTA, and retinal pigment epithelial cells are incubated for 15-30 min; the produced cells are pipetted and transferred to a sterile recovery medium, pipetted, centrifuged; the recovered cells are resuspended in a high-serum growth medium, then the prepared suspension of the retinal pigment epithelial cells is distributed over a culture surfaces and cultivated until the attached cells reach 15-25 % of its area; the suspension with the unattached cells is aspirated, distributed over the fresh culture surfaces and cultivated; and the unattached cells are added with the high-serum growth medium and cultivated until a confluent finish monolayer is produced.

EFFECT: invention allows eliminating damaging action of enzymes and ensuring a lower adhesion cell population to attach on the culture surfaces.

13 cl, 4 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: donor chondrocytes are produced by recovering and grinding donor cartilaginous tissue, carrying out enzymatic treatment, filtration and centrifugation of the prepared chondrocyte suspension and cultivation thereof in a nutrient medium DMEM with added 20 % FBS and perphthorane in amount 15-25 % of total amount of the nutrient medium.

EFFECT: invention allows increasing synthetic activity of chondrocytes in population in vitro, without changing substantially the other cell parameters, such as phenotype, proliferation, ability to colony-formation.

2 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: donor chondrocytes are produced by recovering and grinding donor cartilaginous tissue, carrying out enzymatic treatment, filtration and centrifugation of the prepared chondrocyte suspension and cultivation thereof in a nutrient medium DMEM with added 20 % FBS and perphthorane in amount 15-25 % of total amount of the nutrient medium.

EFFECT: invention allows increasing synthetic activity of chondrocytes in population in vitro, without changing substantially the other cell parameters, such as phenotype, proliferation, ability to colony-formation.

2 dwg, 1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: microorganisms are used to prepare a culture medium, where the microorganisms are cultured on a mineral medium which supports growth of that organism, where essentially all assimilated atoms are isotope-labelled to obtain a labelled biomass. The obtained biomass undergoes autolysis to obtain a labelled autolysate. This biomass can be further extracted with an organic solvent to obtain lipids. The delipidised biomass is hydrolysed to obtain labelled amino acids and other nutrient substances which are used together with the autolysate extracted by lipids and additional components to prepare a culture medium for growing mammal or insect cells in the culture for synthesis of biomolecules. The obtained biomolecule is a mammal membrane protein, particularly in which 20-100% hydrogen atoms in that protein are totally substituted with a 2H isotope.

EFFECT: uniform labelling with stable isotopes enables to determine the three-dimensional structure of a biomolecule such as a mammal membrane protein, for example, using NMR spectroscopy.

30 cl, 4 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to producing versions of group I Poaceae (holy grass) allergen, also can be used either for specific immunotherapy (hyposensitisation) of patients with grass pollen allergy, or for preventive immunotherapy of grass pollen allergies. The produced versions are characterised by Cys41 Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser substitutes in a Phi p1 mature protein sequence. Also, a structure of the allergen versions can be presented with no fragments relevant to amino acid residues 1-6, 1-30, 92-104, 115-119, 175-185 and 213-220 or 1-6, 115-119 and 213-220 as a part of a primary sequence of Phi p1 mature protein.

EFFECT: invention allows producing a version of group I Poaceae allergen characterised lower IgE responsiveness as compared with common wild allergen and substantially maintained responsiveness to T-lymphocytes.

8 cl, 9 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to a method of induced differentiation of embryo stem (ES) cells in neuronal precursor cells. The presented method involves ES cell culture by sowing the ES cells of density approximately 0.5 × 105 - 2 × 105 cells in cm2 and dissociating the ES cells 2 days after sowing. Then, cell aggregates (CA) are formed that involves sampling the cells of high proliferative activity of doubling time within 0 to 24 hours and sowing these cells of density approximately 0.5 × 105 - 5 × 105 cells in ml to form the CAs. Further, the cell aggregates are processed with retinoic acid (RA). Then, the CAs are dissociated to prepare a neuronal precursor cell culture.

EFFECT: presented invention allows preparing substantially homogeneous neuron population wherein practically all neurons belong to the same certain neuronal cell differentiation line, to the same phenotype, cell type and to the same differentiation stage.

26 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to a method of induced differentiation of embryo stem (ES) cells in neuronal precursor cells. The presented method involves ES cell culture by sowing the ES cells of density approximately 0.5 × 105 - 2 × 105 cells in cm2 and dissociating the ES cells 2 days after sowing. Then, cell aggregates (CA) are formed that involves sampling the cells of high proliferative activity of doubling time within 0 to 24 hours and sowing these cells of density approximately 0.5 × 105 - 5 × 105 cells in ml to form the CAs. Further, the cell aggregates are processed with retinoic acid (RA). Then, the CAs are dissociated to prepare a neuronal precursor cell culture.

EFFECT: presented invention allows preparing substantially homogeneous neuron population wherein practically all neurons belong to the same certain neuronal cell differentiation line, to the same phenotype, cell type and to the same differentiation stage.

26 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to a method of induced differentiation of embryo stem (ES) cells in neuronal precursor cells. The presented method involves ES cell culture by sowing the ES cells of density approximately 0.5 × 105 - 2 × 105 cells in cm2 and dissociating the ES cells 2 days after sowing. Then, cell aggregates (CA) are formed that involves sampling the cells of high proliferative activity of doubling time within 0 to 24 hours and sowing these cells of density approximately 0.5 × 105 - 5 × 105 cells in ml to form the CAs. Further, the cell aggregates are processed with retinoic acid (RA). Then, the CAs are dissociated to prepare a neuronal precursor cell culture.

EFFECT: presented invention allows preparing substantially homogeneous neuron population wherein practically all neurons belong to the same certain neuronal cell differentiation line, to the same phenotype, cell type and to the same differentiation stage.

26 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: there is offered a method for producing adult stem cells with multipotent properties that involves recovering thereof from human brown adipose tissue and cultivating in a nutrient medium containing N-acetyl-L-cysteine, as well as a method for long-term maintaining these cells in the undifferentiated condition due to formation of spheres in a CORM-2-containing medium. The adult stem cells produced by the offered method are characterised by high proliferation rate, positive CD73, CD90, CD29, CD44 and CD 105 immune response, negative CD33, CD34, CD45, CD4, CD31, CD62p, CD14 and HLA-DR immune response and ability to differentiate in cells of mesodermal origin. Particularly, disclosed is a possibility of producing nervous cells, cartilaginous cells, osteogenous cells, adipose cells and insulin-producing pancreatic cells of said adult stem cells under the invention.

EFFECT: method improvement.

12 cl, 15 dwg, 2 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: solution of viable breast cells after enzymatic degradation in a collagenase solution of analysed tissue at temperature 37°C for 30-35 minutes, are applied on a biomaterial of a native form of hyaluronic acid, stored at room temperature and constant humidity 30-50 %, and cell viability is controlled by discoloration of cell monolayer.

EFFECT: invention allows producing a viable cell monolayer and providing their vital activity for 2-3 hours.

1 tbl, 2 dwg

FIELD: medicine.

SUBSTANCE: eyeball is enucleated in autopsy, washed in an alcoholic solution and in Ca2+ Mg2+ -free Hanks' solution with an antibiotic added; an anterior segment of an eyeball is removed along a dentate line, a vitreous body and a neutral retina are separated from a pigment epithelium; an eyecup is filled with Ca2+ Mg2+ -free Hanks' solution with EDTA, and retinal pigment epithelial cells are incubated for 15-30 min; the produced cells are pipetted and transferred to a sterile recovery medium, pipetted, centrifuged; the recovered cells are resuspended in a high-serum growth medium, then the prepared suspension of the retinal pigment epithelial cells is distributed over a culture surfaces and cultivated until the attached cells reach 15-25 % of its area; the suspension with the unattached cells is aspirated, distributed over the fresh culture surfaces and cultivated; and the unattached cells are added with the high-serum growth medium and cultivated until a confluent finish monolayer is produced.

EFFECT: invention allows eliminating damaging action of enzymes and ensuring a lower adhesion cell population to attach on the culture surfaces.

13 cl, 4 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: donor chondrocytes are produced by recovering and grinding donor cartilaginous tissue, carrying out enzymatic treatment, filtration and centrifugation of the prepared chondrocyte suspension and cultivation thereof in a nutrient medium DMEM with added 20 % FBS and perphthorane in amount 15-25 % of total amount of the nutrient medium.

EFFECT: invention allows increasing synthetic activity of chondrocytes in population in vitro, without changing substantially the other cell parameters, such as phenotype, proliferation, ability to colony-formation.

2 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: donor chondrocytes are produced by recovering and grinding donor cartilaginous tissue, carrying out enzymatic treatment, filtration and centrifugation of the prepared chondrocyte suspension and cultivation thereof in a nutrient medium DMEM with added 20 % FBS and perphthorane in amount 15-25 % of total amount of the nutrient medium.

EFFECT: invention allows increasing synthetic activity of chondrocytes in population in vitro, without changing substantially the other cell parameters, such as phenotype, proliferation, ability to colony-formation.

2 dwg, 1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: microorganisms are used to prepare a culture medium, where the microorganisms are cultured on a mineral medium which supports growth of that organism, where essentially all assimilated atoms are isotope-labelled to obtain a labelled biomass. The obtained biomass undergoes autolysis to obtain a labelled autolysate. This biomass can be further extracted with an organic solvent to obtain lipids. The delipidised biomass is hydrolysed to obtain labelled amino acids and other nutrient substances which are used together with the autolysate extracted by lipids and additional components to prepare a culture medium for growing mammal or insect cells in the culture for synthesis of biomolecules. The obtained biomolecule is a mammal membrane protein, particularly in which 20-100% hydrogen atoms in that protein are totally substituted with a 2H isotope.

EFFECT: uniform labelling with stable isotopes enables to determine the three-dimensional structure of a biomolecule such as a mammal membrane protein, for example, using NMR spectroscopy.

30 cl, 4 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to producing versions of group I Poaceae (holy grass) allergen, also can be used either for specific immunotherapy (hyposensitisation) of patients with grass pollen allergy, or for preventive immunotherapy of grass pollen allergies. The produced versions are characterised by Cys41 Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser substitutes in a Phi p1 mature protein sequence. Also, a structure of the allergen versions can be presented with no fragments relevant to amino acid residues 1-6, 1-30, 92-104, 115-119, 175-185 and 213-220 or 1-6, 115-119 and 213-220 as a part of a primary sequence of Phi p1 mature protein.

EFFECT: invention allows producing a version of group I Poaceae allergen characterised lower IgE responsiveness as compared with common wild allergen and substantially maintained responsiveness to T-lymphocytes.

8 cl, 9 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to a method of induced differentiation of embryo stem (ES) cells in neuronal precursor cells. The presented method involves ES cell culture by sowing the ES cells of density approximately 0.5 × 105 - 2 × 105 cells in cm2 and dissociating the ES cells 2 days after sowing. Then, cell aggregates (CA) are formed that involves sampling the cells of high proliferative activity of doubling time within 0 to 24 hours and sowing these cells of density approximately 0.5 × 105 - 5 × 105 cells in ml to form the CAs. Further, the cell aggregates are processed with retinoic acid (RA). Then, the CAs are dissociated to prepare a neuronal precursor cell culture.

EFFECT: presented invention allows preparing substantially homogeneous neuron population wherein practically all neurons belong to the same certain neuronal cell differentiation line, to the same phenotype, cell type and to the same differentiation stage.

26 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to a method of induced differentiation of embryo stem (ES) cells in neuronal precursor cells. The presented method involves ES cell culture by sowing the ES cells of density approximately 0.5 × 105 - 2 × 105 cells in cm2 and dissociating the ES cells 2 days after sowing. Then, cell aggregates (CA) are formed that involves sampling the cells of high proliferative activity of doubling time within 0 to 24 hours and sowing these cells of density approximately 0.5 × 105 - 5 × 105 cells in ml to form the CAs. Further, the cell aggregates are processed with retinoic acid (RA). Then, the CAs are dissociated to prepare a neuronal precursor cell culture.

EFFECT: presented invention allows preparing substantially homogeneous neuron population wherein practically all neurons belong to the same certain neuronal cell differentiation line, to the same phenotype, cell type and to the same differentiation stage.

26 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to a method of induced differentiation of embryo stem (ES) cells in neuronal precursor cells. The presented method involves ES cell culture by sowing the ES cells of density approximately 0.5 × 105 - 2 × 105 cells in cm2 and dissociating the ES cells 2 days after sowing. Then, cell aggregates (CA) are formed that involves sampling the cells of high proliferative activity of doubling time within 0 to 24 hours and sowing these cells of density approximately 0.5 × 105 - 5 × 105 cells in ml to form the CAs. Further, the cell aggregates are processed with retinoic acid (RA). Then, the CAs are dissociated to prepare a neuronal precursor cell culture.

EFFECT: presented invention allows preparing substantially homogeneous neuron population wherein practically all neurons belong to the same certain neuronal cell differentiation line, to the same phenotype, cell type and to the same differentiation stage.

26 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to surgery, and can be used in treating patients with secondary lymphedema of upper extremities. That is ensured by the introduction of autolymphocytes extracorporally processed with Roncoleukin and dissolved in normal saline in subcutaneous fat of an injured extremity in a projection of lymph node basins along a medial and lateral surface of forearm. The solution is introduced in 20 points by 0.5 ml containing 20 to 30 million cells per each point on the average. The procedures are thrice-repeated every 72 hours.

EFFECT: method allows to intensify lymphatic outflow from the injured extremity, to reduce paravasal inflammations of subcutaneous fat due to stimulating cell components of the immune system.

8 tbl, 1 ex

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