RU2523563C1 - Bone marrow technology from dead donors with beating and non-beating heart - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: wings of ilium are punctured in an anterior and posterior one-third of the wings with two trocars being inserted into each wing. The bone marrow (BM) is collected by simple aspiration, aspiration irrigation or a combination thereof at an underpressure of 0.6 Atm with using a device. The bone marrow preparation device comprises a disposable multi-channel closed system, an aspiration collection unit and a perfusion unit. The group of inventions also refers to a method for assessing the prepared bone marrow. The effect is ensured by automatic control of myeloaspiration by preparing a biological material with using a special designed device for the bone marrow collection.

EFFECT: using the given method for preparing the bone marrow provides preparing the sterile bone marrow rich in viable multipotent mesenchymal stromal and hemopoietic progenitor cells.

7 cl, 1 ex, 1 dwg, 1 tbl

Description

The invention relates to medicine and can be used for cell therapy in patients and victims in order to improve reparative processes.

In the modern sense, a stem cell is an immature cell capable of self-renewal and differentiation into specialized cells of the body. Being a universal stable module, stem cells can create resistant sprouts of new healthy tissue in diseased organs. The significance of this approach for the restoration or restoration of healthy tissues in humans instead of diseased or lost ones was determined by Order of the Minister of Health of the Russian Federation No. 325 of 07.25.2003 “On the Development of Cellular Technologies in the Russian Federation”.

Most often in clinical practice, hematopoietic (capable of differentiating into blood cells) and mesenchymal (precursors of connective, bone, muscle, fat, cartilage tissue cells) progenitor cells are used. When evaluating the effectiveness and standardizing the protocols of cell therapy, the term “therapeutic dose” is used. “Therapeutic dose” - the optimal number of progenitor cells, allowing to achieve the expected effect. For systemic administration, progenitor cells are usually used in an amount of 1-2 * 10 ⁶ / kg of patient weight. When applied topically, the dose of cells depends on the pathology and prevalence of the process.

A rich source of progenitor cells of both types is human bone marrow (CM). In the process of obtaining stem cells from KM, the following steps are performed: collection of KM, its purification, fractionation with the release of progenitor cells. The resulting cellular material can be cultured, modified, stored, or used in clinical practice.

KM donors are collected from the ilium wings (CPC) with a syringe through a bone marrow needle. In order to prevent coagulation of the trocar aspirate, the collection device and the storage tank are treated with an anticoagulant. The skin, the underlying soft tissues are pierced with a trocar, the spongy bone is punctured. Creating a vacuum in the device connected to the trocar, they collect the CM. At once, up to 5 ml of KM is aspirated, after which the trocar position in the bone is changed. After aspiration, 15-20 ml of KM change the puncture point of the bone. The procedure is repeated until the required volume of aspirate is obtained. Since the aspirate is significantly different in cellularity from native KM, since it is significantly diluted with blood, washing progenitor cells from the spongy bone, then to obtain the necessary therapeutic dose, myeloaspiration is carried out in a volume of up to 1500 ml.

The described technique is most often used in practical health care. However, the routine use of the standard myeloaspiration technique to obtain CM progenitor cells suitable for transplantation is hampered by the inherent disadvantages of the method. Performing a large number of punctures with a trocar of soft tissues, bones, to obtain the required volume of CM, requires an anesthesia to the donor or spinal anesthesia. A large volume of intraoperative trauma causes a high risk of complications in the postoperative period [Impact of stem cell donation modality on normal donor quality of life: a prospective randomized study, GA Kennedy et al., 2003]. The amount of aspirate collected is equivalent to blood loss, which requires adequate infusion-transfusion therapy. High requirements for the somatic state cause a shortage of volunteer donors and contraindications to autologous donation in some groups of patients.

According to the literature, the source for obtaining a large number of progenitor cells is the CM of corpse donors [Cadaveric bone marrow and spleen cells for transplantation, G Soderdahl et al., 1998]. Existing methods for obtaining KM from corpse donors are conventionally divided into two groups: those involving extraction of bones containing KM from the body, followed by their processing to obtain progenitor cells and based on aspiration of biomaterial [Methods for obtaining the maximum possible amount of bone marrow and blood from corpses / П .M. Medvedev, A.A. Rakov, G.A. Pafomov and others: Method. rivers - Leningrad, 1982. - 25 p.].

In accordance with the Instruction for the Preparation and Preservation of the Posthumous Bone Marrow of the Ministry of Health of the USSR of 1989, myeloaspiration in a corpse donor is carried out in a manner similar to that used in volunteer donors with a vacuum of no more than 0.4 Atm. However, the effectiveness of this method in corpse donors, as a rule, is lower than in healthy ones, which is associated with changes in the rheological and dynamic properties of blood that occur when cardiac arrest stops. Posthumous hemocoagulation leads to thrombosis of the blood vessels of the bone and prevents the progenitor cells from leaching from the blood cells. By partially turning off the bone from the blood circulation, it is possible to collect concentrated KM, however, a large number of progenitor cells are not collected. With the complete cessation of blood flow in the bone tissue, the vacuum created by aspiration makes the collection procedure not effective. The solution to this problem is to resolve the vacuum created by aspiration in bone tissue by connecting to the area containing the CM, an external source of washing fluid.

The closest technical solution adopted for the prototype is the method approved for working with corpses [Methods for obtaining the maximum possible amount of bone marrow and blood from corpses / P.M. Medvedev, A.A. Rakov, G.A. Pafomov, etc. : Method. rivers - Leningrad, 1982. - 25 p.].

In accordance with the method of “Aseptic harvesting of bone marrow from corpses in the field” [Methods for obtaining the maximum possible amount of bone marrow and blood from corpses / P.M. Medvedev, A.A. Rakov, G.A. Pafomov, etc. : Method. rivers - Leningrad, 1982. - 25 pp.], Myeloaspiration is performed through a double-lumen trocar with a bone marrow capture and transplantation apparatus (AVIT-1). One channel is used for aspiration, and the other is perfusion with a working solution. The resulting biomaterial assesses the volume, content of nucleated cells and their viability. The main disadvantage of this method is the aspiration to a greater extent of the working solution than KM [G.I. Kogut. Some methodological features of obtaining cadaveric bone marrow for clinical use // Clinical Surgery. - 1979. - No. 3. - S. 39-41]. The disadvantages of the method include the need, after collecting 15-30 ml of KM, to change the installation point of the trocar, which increases the complexity of the method. The disconnection of the AVIT-1 apparatus from the trocar when the puncture point of the bone changes, makes the system open and increases the likelihood of infection of the biomaterial by pathogenic agents.

Thus, in order to obtain a sterile cell rich in viable multipotent mesenchymal stromal and hematopoietic progenitor CM cells, a new method is required from donor corpses that allows collecting by aspiration, aspiration-perfusion, and combining them.

Achievable technical result is the provision of a sterile rich harvest with viable multipotent mesenchymal stromal and hematopoietic progenitor CM cells.

The result is achieved by:

- maintaining a closed and full-blooded circulatory system by collecting KM before blood, organs, tissues are harvested, or by compressing the aorta and inferior vena cava during organ donation prior to perfusion of organs with a preservative solution, thus preserving the necessary amount of blood for performing myeloexfusion, eliminating contact KM with environment and preserving solution. Myeloaspiration is essentially washing out progenitor cells from the cells of the spongy bone with liquid blood (aspiration), and a working solution (aspiration-washing). In the case of non-clamping of the vessels, when organs are removed, blood is poured into the abdominal cavity, becomes infected, the gaps of the vessels gap, not allowing the necessary tension to be created in the bone, providing the infected blood with the bone marrow,

- the simultaneous installation of trocars in the bone, thus ensuring sterility of the material, since subsequently there is no opening of the contour for rearranging the trocars,

- automation of myeloaspiration, by preparing biomaterial with a specially developed device for collecting KM, including a disposable multichannel sterile closed system (OMZS), surgical suction, volumetric pump, disposable storage tank, system for volumetric pump, working solution, which ensure the extraction of KM by aspiration, aspiration -washing, as well as their combination during discharge up to 1.0 Atm, with predetermined and real-time controlled discharge and perfusion rate of the myelin region Oaspiration, without opening and unsterilizing the circuit during the conversion of methods,

- aspiration of the biomaterial with a discharge of 0.6 Atm, which provides a significant increase in the amount of biomaterial being procured, its cellularity, while not significantly increasing the number of damaged cells.

The method is as follows.

Procurement of CM and blood from corpses is carried out on the basis of the federal law of the Russian Federation dated 21.11.2011 No. 323-ФЗ "On the basics of protecting the health of citizens in the Russian Federation", the federal law of the Russian Federation dated 22.12.1992 No. 4180-1 "On organ transplantation and ( or) human tissue. " In accordance with federal law, organs and tissues are removed from donor corpses with a beating heart (in a state of brain death) and not beating heart.

Corpse-beating donors with a beating heart - donors with ascertained death of the brain against the background of preserved cardiac activity, connected to a system that ensures the proper level of tissue oxygenation.

Corpse donors with a non-beating heart - donors with ascertained biological death.

1. Assessment of the suitability of a corpse donor for the collection of CM.

It is carried out in accordance with normative legal acts in force on the territory of the Russian Federation.

When evaluating a corpse donor, one examines the anamnesis of life, illness, a description of the treatment, the conditions of occurrence and state of death, conduct an external examination. A donor corpse is not suitable for myeloexfusion in case of lack of blood circulation for more than 6 hours, detection of oncological, infectious (in the last 6 months), autoimmune diseases, liver cirrhosis, malaria (in the presence of seizures in the last 3 years), gastric ulcer and 12 duodenal ulcer (exacerbations in the last 6 months), acute or exacerbations of chronic inflammatory diseases of any localization (within the last month), skin diseases, mental illnesses, drug addiction, death from trauma accompanied by penetration conductive injuries, massive blood loss, poisoning (except poisoning by ethyl alcohol), water asphyxia or vomitus.

In the absence of a history or history of data-contraindications to donation, the donor-corpse is considered suitable for myeloexfusion.

2. Preparation of the donor for the collection of CM.

The preparation of a donor for collecting CT depends on the tactics and volume of harvesting organs and tissues. The main goal is to provide the ability to collect CM. The main tasks are determining the sequence and methods of taking tissues and organs, ensuring the preservation of blood in the vessels supplying the CCP.

When harvesting from a corpse donor, only CMs are placed in the “on the stomach” position. Landmarks are determined: crests of the wings of the ilium, Michaelis rhombus, the intersection of the rear axillary lines with the crests of the CPC. Antiseptic treatment of the skin of the lumbar region, buttocks, corresponding lateral surfaces of the body is carried out. Sterile operating clothes limit the surgical field above the CPC along the back and side surfaces of the body.

For myeloaspiration from a donor-corpse to the operation of harvesting organs and other tissues, it is placed in the “on the back” position. Landmarks are determined: crests of the wings of the ilium, anterior-superior awns, the intersection of the middle axillary lines with the crests of the CPC. Antiseptic treatment of the skin of the abdomen, upper third thighs, and the corresponding lateral surfaces of the body is performed. Sterile operating linen limit the surgical field over the wings of the ilium (CPC) along the front and side surfaces of the body.

When collecting KM during or after organ transplantation, CPC is punctured from the abdominal cavity through the laparotomy wound, therefore, donor placement, formation of the surgical field, and access to the abdominal cavity are performed by specialists performing organ explantation. To maintain a sufficient amount of blood in the vessels of the ileum and lower extremities to perform myeloexfusion, to prevent the preservation of the preservation solution in it, immediately before the organs are perfused, they overlap the aorta and the inferior vena cava distal to the renal vessels by applying clamps or ligatures.

Next, prepare a device for collecting KM.

3. Preparation of the device for collecting KM.

The device allows sterilized CM to be harvested using simple aspiration and aspiration-washing methods, as well as combining them without opening the system loop.

A device for collecting KM includes:

1. Disposable multi-channel closed system for collecting KM (OMZS, Figure 1) - nodes and plastic channels connected in a special order. It provides sterility of biomaterial, simultaneous collection of CM through several points of bone puncture (up to 4) using simple aspiration, aspiration-washing, conversion of one method to another, sampling during harvesting without opening the circuit, directed current of myeloaspirate, working solution. Includes blocks:

1. The aspirate removal unit consists of:

1.1. 1st node of connection to the aspiration-accumulation module - Luer-Lock male port

1.2. 1st channel connection node - Y-shaped tee

1.3. 2 nodes of drug administration and sampling - injection port

1.4. 3 nodes of overlapping channels - plastic clip

1.5. 5 channels connecting nodes - a PVC tube with an external diameter of 10 mm, a wall thickness of 1 mm.

2. 2 blocks connecting to trocars consisting of:

2.1. 2 nodes connecting to trocars - Luer-Lock male port

2.2. 2 nodes connecting channels - Y-shaped tee

2.3. 2 nodes of overlapping channels of nodes connecting to trocars - plastic clip

2.4. 2 channels of nodes connecting to trocars - a PVC tube with an external diameter of 10 mm, a wall thickness of 1 mm.

2.5. Channel 1 main - PVC pipe with an external diameter of 10 mm, a wall thickness of 1 mm.

2.6. 1st node of overlapping the main channel - plastic clip

3. The supply unit of the working solution consists of:

3.1. 1st node for connecting to the perfusion module - Luer-Lock female port

3.2. 1st channel connection node - Y-shaped tee

3.3. 3 nodes of overlapping channels - plastic clip

3.4. 3 channels connecting nodes - a PVC tube with an external diameter of 10 mm, a wall thickness of 1 mm.

2. The aspiration-accumulation module includes a connected storage tank and surgical suction. Provides sterility of biomaterial, directed current of myeloaspirate, its accumulation.

- Storage tank - a sterile tank, with ports for inflow and to create a vacuum, preserving the vacuum and sterility of the biomaterial when discharged to 1.0 Atm.

- Surgical suction - an electric device that creates and maintains a vacuum of up to 1.0 Atm.

3. The perfusion module includes a volumetric pump with a system, a container with a working solution. Provides sterility of biomaterial, directed current of working solution.

- Volumetric pump - an electric device that creates a positive pressure that controls the rate of infusion.

- The system for the volumetric pump is sterile, standard, for the volumetric pump used, a system having a port for connecting to the patient in the form of a Luer-lock male, a channel blocking unit.

- A container with a working solution is a container containing a sterile isotonic solution mixed with an anticoagulant.

The collection and preparation for operation of the device is performed while maintaining the sterility of the components:

1. Weigh the storage capacity.

2. Prepare a working solution - the isotonic solution is mixed with heparin, the final concentration of anticoagulant was 25 million units / liter.

3. Prepare OMZS - close all nodes overlapping channels. The node for connecting to the perfusion module (Figure 1. 3.1) is connected to the port for connecting to the patient of the system for volumetric pump (Luer-lock male). The connection node to the aspiration-accumulation module (Figure 1. 1.1) is connected to the inflow port of the storage capacity.

4. Prepare the perfusion module - the system for the volumetric pump is installed in the volumetric pump, connected to the tank with the working solution. On the system close the channel overlap node.

5. Prepare the module of aspiration-accumulation - a surgical suction is connected to the port to create a vacuum of the storage capacity.

6. Fill the device with a working solution - turn on the volumetric pump of the perfusion module and set it to a perfusion speed of 800 ml / hour. Open the system channel overlap node for the volumetric pump, the nodes on the OMZS channels (Figure 1. 1.4, 2.3, 2.6, 3.2). The working solution fills the channels of the device. When the working solution reaches the node connecting to the trocar (Figure 1. 2.1), on the channel going directly to the node (Figure 1. 2.4), close the channel blocking node (Figure 1. 2.3). They fill the OMZS with a working solution, close all nodes of the overlapping of the OMZS channels, and then turn off the volumetric pump.

7. Create a working vacuum in the device - turn on the electric pump, configure it to create and maintain a pressure of 0.6 Atm.

After collecting the device and preparing it for work, they proceed to the puncture of the CPC and myeloaspiration.

4. Puncture of the wings of the ilium and the collection of KM.

For simultaneous harvesting of CMs from several bone sites by methods of simple aspiration, aspiration-washing, 2 trocars are installed in each CPC. The anterior and posterior third of the PDA are punctured. The puncture method is chosen depending on the donor placement, the technique of the operation performed before, after, parallel to myeloexfusion:

- Percutaneous puncture of the CPC in the position of the donor "on the stomach." To puncture the posterior third of the CPC, a skin puncture is performed at a point 5 cm outward from the lateral angle of the Michaelis rhombus. The trocar is oriented perpendicular to the skin, carried through soft tissue until it stops in the bone. The periosteum, a compact substance, is pierced, the spongy substance of the bone is punctured. To puncture the anterior third of the CPC, a skin puncture is performed at the point of palpation of the edge of the crest of the CPC along the posterior axillary line. The trocar is carried through soft tissues until it stops in the crest of the ilium. The tip of the trocar round the edge of the crest. Sliding the tip along the inner surface of the PDA, the tool is advanced in the direction of the pubic joint until it stops. The periosteum, a compact substance, is pierced, the spongy substance of the bone is punctured.

- Percutaneous puncture of the CPC in the position of the donor "on the back." To puncture the posterior third of the CPC, a skin puncture is performed at the point of palpation of the edge of the crest of the CPC along the mid-axillary line. The trocar is carried through soft tissue all the way to the bone. The tip of the trocar round the edge of the crest. Sliding the point along the inner surface of the PDA, the tool is advanced in the direction of the ileo-iliac joint until it stops. The periosteum, a compact substance, is pierced, the spongy substance of the bone is punctured. For puncture of the anterior third of the iliac wing, the anterior superior spine is palpated, they are shifted 2 cm laterally along the crest to obtain a point at which soft tissues are punctured. The trocar is installed on the iliac crest perpendicular to the plane, periosteum, compact substance are punctured, spongy substance is punctured.

- Transabdominal puncture of the CPC in the position of the donor "on the back." Transabdominally CPC is punctured through the laparotomy wound. In the abdominal cavity, the borders of the ilium, large lumbar muscles, ureters, large vessels of the iliac region, the anterior superior awns are determined. To puncture the posterior third of the CPC with a trocar, mark a point along the outer edge of the psoas major muscle at the level of the promontorium cape. At this point, the iliac muscle is pierced with a trocar. The tool is oriented perpendicular to the surface of the punctured muscle, it is passed through the soft tissue until it stops into the bone. Periosteum, cortical layer of bone are punctured, spongy substance is punctured. For puncture of the anterior third of the CPC, a soft tissue trocar puncture is performed at the point of palpation of the anterior superior spine. The tool is carried out along the inner surface of the PDA vertically down to the stop. The periosteum, the compact part of the bone is pierced, the cancellous bone is punctured

As they are installed, the nodes of connection to the OMZS trocars are connected to the trocars (Figure 1, 2.1), on which the channels overlapping nodes are opened (Figure 1, 2.3). The collection of CM by simple aspiration begins after the installation of the first trocar, by aspiration-leaching after installing two trocars in one bone.

5. Collection of KM device.

Collecting KM is carried out by the method of simple aspiration, aspiration-washing, or their combination with a discharge of 0.6 Atm. The indicated vacuum is greater than that used for myeloaspiration (0.4 Atm), however, it allows to increase the collection efficiency (Table 1). A pressure of 0.6 Atm causes the collection of a larger volume of myeloaspirate with higher cellularity, thereby increasing the efficiency of myeloaspiration by 2 times, slightly increasing the number of dead cells.

Table 1 Quantitative characteristics of CM samples prepared under different conditions Mode Volume Nucleated Cells GSK (CD45 ^low CD34 ⁺ )

suction ml Concentration, cells / µl Total, × 10 ⁶ Dead cells,% Concentration% Total, × 10 ⁶ 0.4 atm (n = 5) 148 12/24 3570 14.6 0.69 24.54 0.65 atm (n = 11) 190 35.58 6761 20.79 0.75 51.02 0.9 atm (n = 6) 98 64.94 6364 40 1,02 64.68

The method of myeloaspiration is determined depending on the blood supply conditions of the area containing KM:

- The collection of CM by the simple aspiration method is carried out, if blood flow to the bone through natural anatomical formations is possible in the vessels of the ileum and lower extremities, then the method of simple aspiration is preferred. Simple aspiration is a method of collecting KM by creating a vacuum and washing the cells from the cells of the spongy substance with their own blood from blood vessels supplying the bone. Collected progenitor cells are in the most physiological conditions.

For the preparation of CM by simple aspiration, nodes are opened that overlap the channels of the aspirate removal units (Figure 1. 1.4), connections to trocars (Figure 1. 2.3, 2.4). In the process of collecting control the vacuum suction. Myeloexfusion is carried out for 60 minutes.

- The collection of CM by the method of aspiration-washing is performed in the absence of blood flow through the vessels supplying the bone. Aspiration-washing is a method of collecting KM by creating a vacuum and perfusion of the solution from an external source in the area containing KM cells.

For the preparation of CM by the aspiration-wash method, open the nodes of overlapping the channels of the aspirate removal units (Figure 1. 1.3), supply the working solution (Figure 1. 3.2), on the channels of the nodes connecting to the trocars (Figure 1. 2.3). In the process of collecting control the vacuum suction, the perfusion rate of the volumetric pump 800 ml / hour. Myeloexfusion is carried out for 60 minutes.

- Collecting CM by combining methods is optimal for most cases. When combining methods, first perform a simple aspiration method to significantly reduce its effectiveness, then convert to the aspiration-rinse method.

To make the transition from one method to another (conversion), close the nodes of overlapping the main channels (Figure 1. 2.6) of the unit for connecting to trocars, open the nodes of overlapping channels on the channels of the block for supplying the working solution (Figure 1. 3.2). The volumetric pump is retuned to a perfusion rate of 800 ml / hour. For the conversion of aspiration-washing into simple aspiration, the volumetric pump is turned off and the described operations are performed in the reverse order.

To complete the collection of CMs, if it was turned on, the volumetric pump is turned off, the nodes for blocking the channels of the solution supply unit (Figure 1. 3.2), the unit for connecting to trocars (Figure 1. 2.3, 2.6), and the biomaterial removal unit (Figure 1. 1.1). The surgical suction is adjusted to the minimum possible for the discharge device, after which it is turned off. The storage capacity is disconnected from OMZS, surgical suction, the connection ports are tightly closed. The trocars are removed. Skin punctures are sutured.

6. Assessment of myeloaspirate.

The volume of biomaterial is determined by weighing the storage tank with a myeloaspirate and subtracting the empty tank weight from the obtained value. The resulting value is expressed in ml.

A sample is taken from the myeloaspirate to assess:

- The number of nucleated cells. The criterion takes into account all myeloaspirate cells except red blood cells and platelets. It is expressed in the number of cells per unit volume. The indicator allows you to indirectly judge the quality of the biomaterial, as it does not reflect the actual number of progenitor cells.

- The number of hematopoietic stem cells (HSC, CD45 ^low CD34 +) is a new criterion for the assessment of CM of corpse donors, taking into account the cells of the hematopoietic precursors. Relative and absolute values are determined. The criterion takes into account only one type of cell and allows a more accurate assessment of the quality of the biomaterial.

- The number of multipotent mesenchymal stromal cells (MMSC, CD45-CD34-CD105 + CD90 +) is a new criterion for the assessment of CM, taking into account the mesenchymal progenitor cells. The relative and absolute values are determined. The criterion takes into account only one type of cell and allows a more accurate assessment of the quality of the biomaterial.

- The number of dead cells (7AAD) - a new criterion for assessing CM, reflecting the proportion of damaged cells. When a cell dies, its membrane is damaged, the dye penetrates and specifically binds to DNA. It is determined as a percentage of the total number of cells. The criterion takes into account only dead cells and allows a more accurate assessment of the quality of the biomaterial.

After determining the described indicators, the quality of the biomaterial is evaluated:

- “Good” quality in the case of collection of nucleated cells more than 18.1 × 10%, HSC more than 90.1 × 10 ⁶ , MMSC more than 120.1 × 10 ⁶ , dead cells less than 19%;

- “Satisfactory” quality in the case of collection of nucleated cells 10-18 × 10%, HSCs more than 40.1-90 × 10 ⁶ , MMSC 60.1-120 × 10 ⁶ , dead cells 20-29%;

- “Poor” quality in the case of collection of nucleated cells less than 10.0 × 10 ⁹ / l, or HSC less than 40 × 10 ⁶ , MMSC less than 60 × 10 ⁶ , dead cells more than 30%.

A biomaterial corresponding to “good” and “satisfactory” quality can be used in clinical practice after confirming its biological safety. Biomaterial of “unsatisfactory” quality is rejected for clinical use, but can be used to obtain progenitor cells for scientific research.

Thus, the proposed new assessment of the biomaterial allows one to reliably determine its quality, since it takes into account four criteria (nucleated cells, HSC, MMSC, dead cells), describes in greater volume the cellular composition of myeloaspirate and its viability.

Example. Collecting CM from a corpse donor with a non-beating heart.

After examination, recognition of suitability, the tissue donor was sent to the operating room.

The donor was placed in a position on the back, landmarks were determined (crests of the wings of the ilium, anterior superior spine, intersection point of the middle axillary line with the crest of the CPC). In accordance with the rules of aseptic and antiseptic prepared the surgical field.

A device for collecting CMs was prepared: the storage capacity was weighed, the working solution was prepared, the OMZS was connected to the storage capacity (Fresenius Medical Care C.A.T.S.) of the aspiration-accumulation module, and a system for the volumetric pump of the perfusion module. The system for the volumetric pump was installed in the device, connected to the tank with the working solution. Turning on the volumetric pump was filled with OMZS working solution. After filling, the volumetric pump was turned off, the surgical suction was turned on and adjusted to a pressure of 0.6 Atm.

The puncture of each CPC was performed at two points. To puncture the posterior third, the skin was pierced with a trocar over the iliac wing crest in the mid axillary line. The working part of the trocar was carried out into the pelvic cavity, spongy substance was punctured. Puncture of the anterior third was performed 2 cm lateral along the ileal crest of the palpation of the anterior superior spine.

After installation of the first trocar, CM collection was started using the simple aspiration method. The connection node to the OMZS trocar was connected to the trocar (Figure 1. 2.1), on the channel of the connection node to the trocar (Figure 1. 2.4), channel overlap nodes were opened on the channels along the shortest path from the trocar to the storage capacity (Figure 1. 1.4, 2.3, 2.6). Through the node for drug administration and sampling (Figure 1. 1.3), through which the CM flowed, 5 million units were introduced into the biomaterial with a syringe heparin. The remaining trocars were sequentially installed and connected. Myeloaspiration was performed for 30 minutes, after which the method was converted from simple aspiration to aspiration-washing. At OMZS, the nodes of overlapping the main channels (Figure 1. 2.6) of the block connecting to the trocars were closed, the nodes of the overlapping of channels (Figure 1. 3.2) of the working solution supply unit were opened. The volumetric pump was turned on, the perfusion rate was adjusted to 800 ml / hour.

KM collection by aspiration-washing was carried out for 30 minutes.

At the end of the CM collection operation, the volumetric pump was turned off. At OMZS, the channel overlapping nodes of the solution supply unit (Figure 1. 3.4), the trocar connection block (Figure 1. 2.3, 2.6), and the biomaterial abstraction block (Figure 1. 1.4) were successively closed. The surgical suction was adjusted to the minimum possible for the discharge device, after which it was turned off. The storage tank was disconnected from OMZS, surgical suction, the connection ports were closed with the attached covers.

OMZS was disconnected from the trocars. The trocars were removed, skin punctures were sutured.

The storage tank with biomaterial was weighed. The difference in weight between the empty and filled storage tanks was considered equivalent to the volume of the biomaterial.

Samples were taken from the myeloaspirate for research. The concentration of nucleated cells, hematopoietic stem cells (HSC, CD45 ^low CD34 +), multipotent mesenchymal stromal cell (MMSC, CD45-CD34-CD105 + CD90 +), and the percentage of dead cells (7AAD) were estimated by flow cytometry in the samples. Given the volume of biomaterial, the concentration of cells, the absolute number of nucleated cells, CCM, MMSC was determined. Assessed the biomaterial. Biomaterial was recognized as good quality.

Claims (7)

1. The method of harvesting bone marrow (KM) from donor corpses with a beating and non-beating heart, consisting in puncture of the bone and collection of KM, characterized in that the ilium wings are punctured in the anterior and posterior third of the wings, installing in each two trocars, the collection of CM is performed by the method of simple aspiration, aspiration-washing or their combination with a discharge of 0.6 Atm, these techniques are carried out using the device according to claim 7. 2. The method according to claim 1, characterized in that the collection of KM is performed prior to the collection of blood, organs, tissues, or clamping of the aorta and inferior vena cava during organ donation operations before organ perfusion with a preservative solution. 3. The method according to claim 1, characterized in that when the donor is “on the stomach”, a CPC puncture is performed, for puncture of the posterior third of the CPC, the skin is punctured at a point 5 cm outward from the lateral angle of the Michaelis rhombus, the trocar is oriented perpendicular to the skin is carried out through the soft tissues until it stops in the bone, the periosteum, compact substance is punctured, bone spongy is punctured, and for puncture of the anterior third of the CPC, a skin puncture is performed at the point of palpation of the edge of the CPC crest along the posterior axillary line, the trocar is passed through the soft Kani fully into the iliac crest, the crest encircle trocar tip edge, the edge sliding along the inner surface of the CCP, the trocar is advanced in the direction of the pubic articulation until it stops, pierce periosteum compact substance punktirujut cancellous bone. 4. The method according to claim 1, characterized in that when the donor is “on the back”, a CPC puncture is performed, for puncture of the posterior third of the CPC, a skin puncture is performed at the point of palpation of the edge of the CPC crest along the mid-axillary line, the trocar is passed through soft tissues until they stop at the bone, they point around the edge of the ridge with the tip of the trocar, sliding the tip along the inner surface of the CPC, they move in the direction of the sacroiliac joint to the stop, pierce the periosteum, compact substance, puncture the spongy bone, and for puncture the anterior the iliac wing reticles palpate the anterior superior spine, laterally shift 2 cm along the crest - receive a point at which soft tissues are punctured, install the trocar on the ilium crest perpendicular to the plane, pierce the periosteum, compact substance, and spongy substance are punctured. 5. The method according to claim 1, characterized in that when the donor is “on the back” CPC is transabdominally punctured, for which CPC is punctured through the laparotomy wound, while for puncture of the posterior third of the CPC, a point is marked on the outer edge of the psoas major muscle with a trocar the cape of the sacrum (promontorium), at this point, the iliac muscle is punctured, the trocar is oriented perpendicular to the surface of the punctured muscle, it is passed through the soft tissue all the way to the bone, the periosteum, cortical layer of the bone is pierced, the spongy substance is punctured in, and for puncture of the anterior third of the CPC, a soft tissue trocar puncture is performed at the point of palpation of the anterior superior spine, the trocar is carried out vertically down to the stop on the inner surface of the CPC, the periosteum, compact part of the bone are punctured, and the cancellous bone is punctured. 6. A method for evaluating harvested bone marrow from corpse donors with a beating and non-beating heart, including sampling from a myeloaspirate sample for evaluation, the sample assesses the number of nucleated cells, the number of hematopoietic stem cells (HSC, CD45 ^low CD34 +), the number of multipotent mesenchymal stromal cells (MMSC, CD45-CD34-CD105 + CD90 +) and the number of dead cells (7AAD), and in the case of collection of nucleated cells more than 18.1 × 10 ⁹ / l, HSC more than 90.1 × 10 ⁶ , MMSC more than 120.1 × 10 ⁶ , dead cells less than 19%, evaluate harvested bone marrow as “good o ”quality; in the case of collection of nucleated cells 10-18 × 10 ⁹ / l, HSCs more than 40.1-90 × 10 ⁶ , MMSC 60.1-120 × 10 ⁶ , dead cells 20-29% - “satisfactory” quality; in the case of collection of nucleated cells less than 10.0 × 10 ⁹ / l, or HSC less than 40 × 10 ⁶ , MMSC less than 60 × 10 ⁶ , dead cells more than 30% - "unsatisfactory" quality. 7. A device for harvesting bone marrow (KM) from donor corpses with a beating and non-beating heart, includes a disposable multi-channel closed system (OMZS) for collecting CM, an aspiration-accumulation module and a perfusion module, and the OMZS consists of interconnected blocks: aspirate removal unit, two trocar connection units, working solution supply unit; the aspiration-accumulation module is connected to the OMZS through the aspirate removal unit, and includes interconnected storage capacity made in the form of a sterile reservoir, with inflow ports and providing the creation of discharge, preservation of vacuum and sterility of the biomaterial when discharged to 1.0 Atm, as well as surgical suction that creates and maintains a vacuum of up to 1.0 Atm; the perfusion module is connected to the OMZS by means of the working solution supply unit and includes a volumetric pump made in the form of an electric device that creates positive pressure and controls the infusion rate, a volumetric pump system having a patient connection port in the form of Luer-lock male and a channel blocking unit, as well as containers with working solution.

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