CN115870109B - Horizontal centrifugal rotor, cell separation device and method - Google Patents
Horizontal centrifugal rotor, cell separation device and method Download PDFInfo
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Abstract
The invention belongs to the technical field of cell separation, and particularly relates to a horizontal centrifugal rotor, a centrifugal device and a cell separation device comprising the horizontal centrifugal rotor, and a cell separation method. The horizontal centrifugal rotor disclosed by the invention comprises: a horizontal centrifugal rotor platform and a centrifugal tube clamp; the horizontal centrifugal rotor platform is provided with a rotation central axis X, and a centrifugal pipe clamp is arranged on the horizontal centrifugal rotor platform; the centrifugal tube clamp is used for placing and limiting the centrifugal tube, so that the centrifugal tube can synchronously rotate with the horizontal centrifugal rotor platform, and the centrifugal tube is provided with a horizontal axis Y after being placed on the centrifugal tube clamp; the centrifuge tube axis Y is perpendicular to the rotation central axis X. The invention adopts the horizontal centrifugal rotor and uses porous materials in a centrifugal tube to realize short-time and one-time centrifugation to obtain PRP preparations with various cell components and concentrations.
Description
Technical Field
The invention belongs to the technical field of cell separation, and particularly relates to a horizontal centrifugal rotor, a centrifugal device and a cell separation device comprising the horizontal centrifugal rotor, and a cell separation method.
Background
Blood contains plasma and blood cells, and the blood cells contain erythrocytes, leukocytes and platelets, wherein:
white blood cells have an effect of inhibiting inflammation, such as skin wounds, and due to high exposure risk of pathogenic microorganisms, it is desirable that the wound be repaired by leukocyte-rich PRP (platelet-rich plasma); in joint repair, however, pathogenic microorganisms are less likely to leak, and thus, a leukocyte-depleted PRP is desirable; platelets are active for repair, and therefore, higher than baseline levels (primary platelet concentrations) are desirable to promote repair, regardless of wound repair or joint repair.
The platelet concentration and activity in whole blood of patients are related to the physical condition of the patient (e.g., elderly patients have low platelet activity, and their PRP therapy usually requires high platelet concentrations for therapeutic effect, and for example, some patients have low platelet concentrations in whole blood and usually require more blood than ordinary people to concentrate to therapeutic concentrations, but large blood volumes can result in great risk to the body of such patients.
Studies have shown that human platelets have diameters of about 2 to 3 microns (μm), red blood cell diameters of about 6 to 9 μm, and white blood cell diameters of about 6 to 19 μm [ K.Fixter, D.J.Rabbolini, B.Valecha, M.C.Morel-Kopp, S.Gabrielli, Q.Chen, W.S.Stevenson, C.M.Ward, mean platelet diameter measurements to classify inherited thrombocytopenias, int J Lab Hematol 40 (2) (2018) 187-195; C.M.Hawkey, P.M.Bennett, S.C.Gascoyne, M.G.Hart, J.K.Kirkwoo, erythrocyte size, number and haemoglobin content in vertebrates, british Journal olHaernatologg 77 (1991) 392-397; M.M.Winer, A.Zeidan, D.Yeheskely-Hayon, L.Golan, L.Minai, E.J.Dann, D.Yelin, in vivo noninvasive microscopy of human leucocytes, sci.rep.7 (1) (2017) 13031. It can be seen that blood cell separation can be based on cell size.
In the prior art, as in chinese patent application CN114099543a (method for preparing prp serum for repairing sports injury), it is disclosed that red blood cells, white blood cells, fibrin and platelet components in whole blood are removed by twice filtration by using porous materials with different sizes, so as to obtain pure serum. The serum obtained by filtration was mixed with platelet rich plasma (the preparation process of which is not described in the invention) to prepare PRP at various concentrations. The technology has complex treatment process and long treatment time, and is difficult to meet the actual clinical requirements. In another example, chinese patent application CN114939345a (platelet-rich plasma preparation apparatus and operation method thereof) also uses porous material to separate cellular components, but uses porous material to remove cellular and protein components in blood such as red blood cells, white blood cells, fibrin, platelets, etc. to obtain serum; the serum is not PRP. In addition, commercial PRP production systems exist on the market, but the amount of blood required is typically large and the processing time is long, as shown in table 1.
Table 1 comparison of commercial PRP preparation System parameters
The prior art mainly aims at obtaining a certain specific component (usually platelet-rich plasma) in blood to separate and prepare cells, and has the defects of large blood volume, long treatment time, no report on a blood cell separation technology for preparing the corresponding component according to the body requirement of a patient, and meanwhile, a cell separation technology with small blood consumption and quick treatment time is also needed.
Disclosure of Invention
In order to overcome the technical defects of large blood sampling amount, multiple times of filtration and separation, complex operation and long separation time of the traditional blood cell separation process, the invention adopts a horizontal centrifugal rotor, and uses porous materials in a centrifugal tube to realize that PRP preparation with various cell components and concentrations is obtained by short-time and one-time centrifugation, and provides a cell separation device and a cell separation method.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a horizontal centrifugal rotor comprising: a horizontal centrifugal rotor platform and a centrifugal tube clamp; the horizontal centrifugal rotor platform is provided with a rotation central axis X, and a centrifugal pipe clamp is arranged on the horizontal centrifugal rotor platform; the centrifugal tube clamp is used for placing and limiting the centrifugal tube, so that the centrifugal tube can synchronously rotate with the horizontal centrifugal rotor platform, and the centrifugal tube is provided with a horizontal axis Y after being placed on the centrifugal tube clamp; the centrifugal tube axis Y is perpendicular to the rotation central axis X; the perpendicular includes a perpendicular intersection or a perpendicular non-intersection.
Further, a centrifugal pipe clamp positioning device and a centrifugal pipe clamp nest are fixedly connected to the horizontal centrifugal rotor platform; the centrifugal tube clamp can be placed in the centrifugal tube clamp nest, and the centrifugal tube clamp positioning device is used for positioning the centrifugal tube clamp at a desired position in the centrifugal tube clamp nest, namely, the position of the centrifugal tube clamp in the centrifugal tube clamp nest can be adjusted in the horizontal direction, and the centrifugal tube clamp positioning device is used for positioning the centrifugal tube clamp at the desired position; the centrifugal pipe clamps are kept relatively static with the horizontal centrifugal rotor platform through the centrifugal pipe clamp positioning device, namely synchronously rotate, and the axis Y of the centrifugal pipe is always perpendicular to the rotation central axis X of the horizontal centrifugal rotor platform.
Further, the centrifugal pipe clamp positioning devices are multiple and uniformly arranged on the horizontal centrifugal rotor platform around the rotation center axis X; the number of the centrifugal pipe clamp sockets is matched with the number and the positions of the centrifugal pipe clamp positioning devices, and the centrifugal pipe clamp sockets are uniformly arranged on the horizontal centrifugal rotor platform. A centrifugal tube clamp can be placed in each centrifugal tube clamp nest, and each centrifugal tube clamp can be provided with at least one centrifugal tube position; when the number of centrifuge tube positions is plural, the centrifuge tube positions may be arranged side by side or may be arranged in a vertically overlapping manner.
Furthermore, in order to facilitate the positioning of the centrifugal pipe clamp by the pipe clamp positioning device, the centrifugal pipe clamp and the pipe clamp positioning device can be respectively provided with a matched positioning protrusion and a positioning hole for positioning connection. Optionally, the centrifugal pipe clamps are provided with positioning holes which are concentrically arranged, the centrifugal pipe clamp positioning device is provided with positioning protrusions matched with the positioning holes, and an included angle alpha between the axis Y of the centrifugal pipe and the centrifugal force direction F can be adjusted through the matching of the positioning protrusions and different positioning holes on the adjacent centrifugal pipe clamps.
Preferably, the included angle α is 0 to 60 degrees. When alpha is 0 degree, the axis Y of the centrifugal tube is parallel to the centrifugal force direction, and at the moment, the axis Y of the centrifugal tube is perpendicularly intersected with the rotation central axis X; when alpha is greater than 0 degrees, the centrifuge tube axis Y is perpendicular to the central axis of rotation X, but does not intersect.
Further, the centrifugal pipe clamp positioning device is fixedly connected to one end, far away from the rotation center axis X, of the horizontal centrifugal rotor platform, and is provided with a fixed point and a free end, wherein the fixed point is positioned on the horizontal centrifugal rotor platform and is connected with the horizontal centrifugal rotor platform; the positioning protrusion is positioned at the free end, the free end can do 90-degree reciprocating rotation around the fixed point towards the centrifugal pipe clamp, and the centrifugal pipe clamp can be connected and released with the horizontal centrifugal rotor platform through the cooperation of the positioning protrusion and the positioning hole.
According to a second aspect of the invention, a centrifugal device is provided, the centrifugal device comprises the horizontal centrifugal rotor, a clamping hole is formed in the center of a horizontal centrifugal rotor platform, and the horizontal centrifugal rotor platform is connected with a rotating shaft of a centrifugal motor through the clamping hole.
In a third aspect of the present invention, there is provided a cell separation apparatus comprising a centrifuge tube and the centrifuge apparatus provided in the second aspect; the centrifuge tube comprises a separation chamber A and a separation chamber B, wherein the separation chamber A and the separation chamber B are separated by a porous material, and the separation chamber B is positioned at one end of the bottom of the centrifuge tube.
Further, the porous material is a high polymer material or an inorganic material; the high polymer material is selected from polycarbonate, polyester, polysulfone and the like, and the inorganic material is selected from diatomite and the like; the pore diameter of the porous material is 1-5 microns.
Optionally, the porous material is fixed to the separation chamber a near the bottom, and the separation chamber a is connected to the separation chamber B through threads.
According to a fourth aspect of the invention, a blood cell separation method is provided, the cell separation device provided by the third aspect of the invention is adopted in the separation method, anticoagulated whole blood is placed in a separation chamber A, a centrifugal tube is placed in a centrifugal tube clamp, an included angle alpha is formed between the axis Y of the centrifugal tube and the direction of centrifugal force F, and the centrifugal tube is centrifuged for 2-5 minutes at 1500-4000 revolutions per minute to realize cell separation.
Further, for a conventional 7mL centrifuge tube, the amount of whole blood is 2-5 mL; centrifuge tubes and centrifuge size implementations can be increased if larger volumes of whole blood are to be processed.
Further, the included angle alpha between the axis Y of the centrifugal tube and the direction of the centrifugal force F can be 0-60 degrees.
When the axis Y of the centrifugal tube is parallel to the centrifugal force direction F (alpha=0), the centrifugal tube is centrifuged for 2-5 minutes at 1500-4000 Revolutions Per Minute (RPM), and the ultra-high platelet concentration PRP can be rapidly obtained; when the axis Y of the centrifugal tube and the direction of the centrifugal force F form a certain angle (0 < alpha is less than or equal to 60 degrees), the centrifugal tube is centrifuged for 2 to 5 minutes at 1500 to 4000 Revolutions Per Minute (RPM), so that the concentration of the platelets in the PRP can be changed within a certain range, and thus, various clinical requirements are met.
The invention has the following advantages:
(1) The invention can prepare the lean white blood cell PRP and the rich white blood cell PRP at the same time by one centrifugation; or, the concentration of the white-lean PRP platelets prepared in the separation chamber B is regulated by changing the included angle (alpha) between the axis Y of the centrifugal tube and the centrifugal force direction F and the rotating speed. However, the traditional process requires two-step centrifugation to obtain the leukocyte-enriched PRP or the leukocyte-depleted PRP;
(2) The invention adopts a small amount of blood (2-5 mL), and PRP with ultra-high platelet concentration (56 times) can be prepared by one centrifugation; whereas the conventional technology requires a large amount of blood to prepare, as in the GenesisCS product of Table 1, PRP with a 10-fold platelet concentration is prepared, and 60mL of blood is collected;
(3) The invention has less blood sampling amount and faster treatment speed (2-5 min), and is obviously superior to the prior separation technology.
Drawings
FIG. 1 is a schematic illustration of the relative positions of centrifuge tube positions and horizontal rotor platform and centrifugal force of the present invention;
FIG. 2 is a schematic diagram showing a comparison of the centrifuge trace of a horizontal rotor centrifuge tube and the centrifuge trace of a fixed angle rotor centrifuge tube according to the present invention;
FIG. 3 is a schematic view of the horizontal centrifugal rotor structure of the present invention;
FIG. 4 is a schematic view of a positioning device for a centrifugal tube clamp according to an embodiment of the invention;
FIG. 5 is a top view of a centrifugal tube clamp according to one embodiment of the invention;
FIG. 6 is a front view of the centrifugal tube clamp of FIG. 5;
FIG. 7 is a schematic diagram of a centrifuge tube separation structure (tube cap not shown) according to one embodiment of the present invention.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Anticoagulation treatment: uniformly mixing whole blood with 0.25g of sodium citrate solution according to the proportion of 10-15:1 by adopting a clinical common anticoagulation method; or directly using BD Vacutainer blood collection tubes with heparin-treated walls to store whole blood.
As shown in fig. 1, the present invention provides a horizontal centrifugal rotor including: a horizontal centrifuge rotor platform 1 and a centrifuge tube clamp (not shown in fig. 1). The horizontal centrifugal rotor platform has a central axis of rotation X perpendicular to the horizontal plane; the centrifugal tube clamp is arranged on the horizontal centrifugal rotor platform 1 and used for placing and limiting the centrifugal tube, the centrifugal tube is placed on the centrifugal tube clamp and provided with a horizontal centrifugal tube axis Y, and the centrifugal tube, the centrifugal tube clamp and the horizontal centrifugal rotor platform can keep synchronous rotation; the centrifuge tube axis Y is perpendicular to the rotation central axis X. The centrifuge tube axis Y' of a conventional fixed angle rotor has an angle θ (θ is not zero, typically 45 °) with the rotational central axis X. Unlike the conventional fixed angle rotor with centrifuge tube forming a certain angle (90-theta) with the horizontal plane, the centrifuge tube is horizontally arranged. As shown in FIG. 2, when the centrifugal tube in the traditional fixed angle rotor centrifuge rotates and is centrifuged, the trajectory S1 of the tube orifice of the centrifugal tube and the trajectory S2 of the tube bottom are not in the same horizontal plane, but in the invention, the axis Y of the centrifugal tube is positioned in the horizontal plane perpendicular to the central axis X of rotation, and the included angle between the axis Y of the centrifugal tube and the centrifugal force F can be adjusted and changed within the range of 0-60 degrees, and the trajectory of the tube orifice of the centrifugal tube and the trajectory of the tube bottom are in the horizontal plane perpendicular to the central axis X of rotation.
In an alternative embodiment, the centrifuge tube clamp is a cylindrical structure with one end open in a conventional fixed angle rotor centrifuge, having a shape that is compatible with centrifuge tubes; the centrifugal pipe clamp can be horizontally arranged inside the horizontal centrifugal rotor platform, and when the centrifugal pipe clamp rotates, the centrifugal pipe clamp and the horizontal centrifugal rotor platform keep synchronously rotating, and the centrifugal pipe can be limited by inserting the centrifugal pipe into the centrifugal pipe clamp, so that the centrifugal pipe can also keep synchronously rotating with the horizontal centrifugal rotor platform. After the centrifuge tube is placed in the centrifuge tube clamp, the centrifuge tube has a horizontal centrifuge tube axis Y and can also be rotated in synchronization with the horizontal centrifuge rotor platform 1. The pipe orifice of the centrifugal pipe faces one end of the rotation central axis X, the centrifugal pipe axis Y and the centrifugal force F have an included angle alpha, and the included angle alpha is 0-60 degrees.
In an alternative embodiment, the centrifugal pipe clamp is fixedly connected with the horizontal centrifugal rotor platform 1, the fixing mode can be an integrated structure, and the centrifugal pipe clamp and the horizontal centrifugal rotor platform are manufactured into an integrated structure through a die; the centrifugal pipe clamps can be fixedly connected to the horizontal centrifugal rotor platform in a bonding, welding or threaded connection mode. After the centrifugal tube clamp is fixed to place the centrifugal tube according to the requirement, the axis Y of the centrifugal tube is parallel to the direction of the centrifugal force F, and the included angle with a specific angle can be included, namely, the included angle alpha=0 degree (the axis Y of the centrifugal tube is parallel to the direction of the centrifugal force F), or 30 degrees, or 35 degrees, or 45 degrees, or 50 degrees, or 60 degrees.
In an alternative embodiment, the centrifugal pipe clamp is detachably connected with the horizontal centrifugal rotor platform, and the horizontal centrifugal rotor platform 1 is further provided with a centrifugal pipe clamp nest 3 and a centrifugal pipe positioning device 4, as shown in fig. 3. The centrifugal pipe clamp 5 is placed in the centrifugal pipe clamp nest 3, the centrifugal pipe clamp 5 is fixed in the centrifugal pipe clamp nest 3 through the centrifugal pipe clamp positioning device 4, and the centrifugal pipe clamp 5 and the centrifugal pipe clamp nest 3 are kept to rotate synchronously with the horizontal centrifugal rotor platform 1.
In an alternative embodiment, as shown in fig. 3-4, by providing a locating boss 6 at the free end of the centrifugal tube clamp locating device 4, the other end of the centrifugal tube clamp locating device 4 is secured to a horizontal centrifugal rotor platform, such as by a screw, with a securing point 7; the free end can be rotated back and forth by 90 degrees around the fixing point 7 in the direction of the centrifugal tube clamp. A plurality of positioning holes 8 are concentrically arranged on the centrifugal tube clamp 5, and the centrifugal tube clamp 5 can be connected and released with the horizontal centrifugal rotor platform 1 by inserting the positioning protrusions 6 into the positioning holes 8. Moreover, the included angle alpha between the axis Y of the centrifugal tube and the centrifugal force direction F can be adjusted by matching the positioning convex 6 with different positioning holes on the adjacent centrifugal tube clamps, and the preferred angle alpha is more than or equal to 0 and less than or equal to 60 degrees. In the structure shown in fig. 3, the number of the centrifugal pipe clamp positioning devices 4 and the centrifugal pipe clamp sockets 3 is 4, the centrifugal pipe clamp positioning devices and the centrifugal pipe clamp sockets 3 are uniformly distributed on the horizontal centrifugal rotor platform 1 around the rotation central axis X, 1 centrifugal pipe clamp 5 can be placed in each centrifugal pipe clamp socket 3, and at least one centrifugal pipe clamp position can be arranged in each centrifugal pipe clamp 5. In one embodiment as shown in fig. 5 and 6 (fig. 6 is a half cross-sectional view of fig. 5), the centrifugal tube clamp 5 is provided with two centrifuge tube positions 2 arranged in an up-down overlapping manner, and two centrifuge tubes can be placed up-down, and the axes of the two centrifuge tube positions are not in the same horizontal plane, but the two axes are parallel and perpendicular to the rotation central axis X, that is, after the centrifuge tubes are placed, the axes of the two centrifuge tubes are parallel and perpendicular to the rotation central axis X.
In an alternative embodiment, the horizontal centrifugal rotor platform 1 is a disc or cylinder structure having a certain thickness. The centrifugal tube nest 3 is a small cylinder recessed on a disc or a cylinder, the diameter of the centrifugal tube nest 3 is smaller than that of the centrifugal tube nest 5, and the position of the centrifugal tube nest 5 can be freely adjusted, namely the included angle alpha between the axis Y of the centrifugal tube and the centrifugal force direction F can be freely adjusted. As shown in fig. 3, holes 9 for conveniently taking the centrifugal tube clamps are arranged on two sides of the centrifugal tube position 2 on the centrifugal tube clamps 5; the holes 9 may be through the centrifugal tube clamp 5 or may be grooves recessed from the upper surface of the centrifugal tube clamp 5 to facilitate placement and removal of the centrifugal tube clamp into and from the centrifugal tube clamp socket.
The invention also provides a centrifugal device, which is compared with the traditional fixed angle rotor centrifugal machine, the centrifugal device provided by the invention is provided with the horizontal centrifugal rotor, and can realize horizontal centrifugation. The center of the horizontal centrifugal rotor platform of the horizontal centrifugal rotor is provided with a clamping hole 10 (shown in figure 3), and the clamping hole is matched with the rotating shaft of the centrifugal motor in size and fixedly connected with the rotating shaft of the centrifugal motor through a screw.
The centrifugal device provided by the invention can be used for centrifugal separation of samples, and in an alternative embodiment, the centrifugal device is a cell separation device for separating blood cell components.
The cell separation device comprises the centrifugal device and the centrifugal tube, wherein the centrifugal tube comprises a separation chamber A, a separation chamber B and a tube cover, and the separation chamber A and the separation chamber B are separated by a porous material.
The separation chamber A and the separation chamber B are two sections of polymer material pipes, such as Polyethylene (PE), polycarbonate (PC) and polypropylene (PP) materials. In an alternative embodiment, the separation chamber A11 and the separation chamber B12 are connected through threads to form a centrifuge tube, as shown in FIG. 7, a first end of the separation chamber B12 is an open end, an outer wall of the open end is provided with external threads, and a second end of the separation chamber B is a closed end, and is a tube bottom of the centrifuge tube; the first end of the separation chamber A11 is a tube orifice of a centrifuge tube, the inner wall of the second end is provided with an inner thread matched with the outer thread of the separation chamber B12, and a porous material 13 is fixed at a position close to the second end of the separation chamber A, and the porous material can be adhered in the separation chamber A.
The pore diameter of the porous material is 1-5 microns, and the porous material can be prepared by adopting a track etching or sintering process.
When the cell separation device is used for cell separation, a centrifuge tube with a porous material after blood is contained is covered with a tube cover and placed on a centrifuge tube clamp, and the centrifuge tube is fixed on a horizontal centrifuge rotor platform for centrifugation, so that separation of different components of cells can be realized. The specific separation steps are as follows:
(1) Placing a proper amount of anticoagulated whole blood into a separation chamber A, wherein a separation chamber B is positioned at one end of the bottom of a centrifugal tube, and a tube cover covers the opening of the centrifugal tube;
(2) And (3) placing the centrifuge tube in the step (1) on a centrifuge tube clamp and fixing the centrifuge tube on a horizontal centrifuge rotor platform, so that the included angle alpha between the axis Y of the centrifuge tube and the direction of the centrifugal force F is 0-60 degrees, and centrifuging for 2-5 minutes at 1500-4000 Revolutions Per Minute (RPM).
In the following examples, the separation chamber A and the separation chamber B are in threaded connection unless otherwise specified; the porous materials are all prepared by track etching; performing horizontal centrifugation by using the horizontal centrifugal rotor shown in fig. 3; anticoagulation treatment: whole blood was stored directly in BD Vacutainer blood collection tubes with heparin-treated walls.
Example 1
The aperture of the polycarbonate porous material in the centrifuge tube is 1 micron, and the capacity of the centrifuge tube is 7mL; after 3 to 4mL of anticoagulated whole blood was placed in separation chamber a, the tube covering the tube orifice was placed in a centrifuge tube clamp and fixed to a horizontal centrifuge rotor platform, with the axis Y of the tube parallel to the direction of centrifugal force F (α=0°), and centrifuged at 3000 Revolutions Per Minute (RPM) for 5 minutes.
The supernatant in separation chamber A was high in platelet concentration with red blood cells removed (1.9 times: the amount of platelets and white blood cells in the sample before centrifugation was 51X 10, respectively) 9 L and 4.9X10 9 The platelet and leukocyte amounts in the centrifuged samples were 98X 10, respectively 9 L and 7.6X10 9 /L)PRP；
The separation chamber B was provided with an ultra-high platelet concentration (56 times the amount of platelets and white blood cells of the sample before centrifugation was 51X 10, respectively) 9 L and 4.9X10 9 The platelet and leukocyte amounts in the centrifuged sample were respectively2859×10 9 L and 0.8X10 9 L) lean leukocyte PRP.
Example 2
The pore diameter of the polyester porous material in the centrifuge tube is 1 micron, and the capacity of the centrifuge tube is 7mL; 3-4 mL of anticoagulated whole blood is placed in a separation chamber A, a centrifuge tube covering a tube orifice is placed in a centrifuge tube clamp and is fixed on a horizontal centrifuge rotor platform, the axis Y of the centrifuge tube is parallel to the direction of a centrifugal force F (alpha=0 degree), the whole blood is centrifuged for 2 minutes at 4000 Revolutions Per Minute (RPM), and the supernatant in the separation chamber A is the high platelet concentration (2.3 times that of the sample platelets and white blood cells before centrifugation are respectively 51 multiplied by 10) 9 L and 4.9X10 9 The platelet and leukocyte amounts in the centrifuged samples were 117X 10, respectively 9 L and 6.3X10 9 L) PRP; the separation chamber B was provided with an ultra-high platelet concentration (47 times the amount of platelets and white blood cells of the sample before centrifugation was 51X 10, respectively) 9 L and 4.9X10 9 The platelet and leukocyte amounts in the centrifuged samples were 2399×10, respectively 9 L and 0.9X10 9 L) lean leukocyte PRP.
Example 3
The aperture of the polycarbonate porous material in the centrifuge tube is 5 microns, and the capacity of the centrifuge tube is 7mL; after 2-3 mL of anticoagulated whole blood was placed in separation chamber a, the tube covering the tube orifice was placed in a centrifuge tube clamp and fixed to a horizontal centrifuge rotor platform, with the axis Y of the tube parallel to the direction of centrifugal force F (α=0°), and centrifuged at 1500 Revolutions Per Minute (RPM) for 5 minutes.
The supernatant in separation chamber A was a high platelet concentration with red blood cells removed (2.1 times the amount of platelets and white blood cells of the sample before centrifugation was 56X 10, respectively) 9 L and 4.5X10 9 The platelet and leukocyte amounts in the centrifuged samples were 119X 10, respectively 9 L and 3.6X10 9 /L)PRP。
The supernatant in the separation chamber B is the high platelet concentration (3.9 times the platelet and white blood cell amount of the sample before centrifugation is 56×10 respectively) 9 L and 4.5X10 9 The platelet and leukocyte amounts in the centrifuged samples were 219X 10, respectively 9 L and 1.7X10 9 L) lean leukocyte PRP.
Example 4
The aperture of the diatomite porous material in the centrifuge tube is 5 micrometers, and the capacity of the centrifuge tube is 7mL; placing 2-3 mL of anticoagulated whole blood in a separation chamber A, placing a centrifuge tube covering a tube orifice in a centrifuge tube clamp and fixing the centrifuge tube on a horizontal centrifuge rotor platform, centrifuging the whole blood for 2 minutes at 2000 Revolutions Per Minute (RPM) with the axis Y of the centrifuge tube parallel to the direction of the centrifugal force F (alpha=0 DEG), wherein the supernatant in the separation chamber A is the high platelet concentration (1.7 times the platelet and leukocyte amounts of a sample before centrifugation are 56×10 respectively) 9 L and 4.5X10 9 After centrifugation, the platelet and leukocyte amounts in the sample were 99X 10, respectively 9 L and 4.7X10 9 /L) leukocyte-enriched PRP. The supernatant in the separation chamber B is the high platelet concentration (2.6 times of the platelet and white blood cell amount of the sample before centrifugation is 56 multiplied by 10 respectively) 9 L and 4.5X10 9 After centrifugation, the amounts of platelets and leukocytes in the sample were 147X 10, respectively 9 L and 2.1X10 9 L) lean leukocyte PRP.
Example 5
The pore diameter of the polysulfone porous material in the centrifuge tube is 2 microns, and the capacity of the centrifuge tube is 7mL; after 3 to 4mL of anticoagulated whole blood was placed in separation chamber a, the centrifuge tube port was capped and placed in a centrifuge tube clamp and fixed to a horizontal centrifuge rotor platform, centrifuge tube axis Y was parallel to the direction of centrifugal force F (α=0°), and centrifuged at 3500 Revolutions Per Minute (RPM) for 5 minutes.
The separation chamber A had a high platelet concentration (1.7 times the amount of platelets and white blood cells in the sample before centrifugation was X10, respectively) 9 L and 4.6X10 9 The platelet and leukocyte amounts in the centrifuged samples were 57X 10, respectively 9 L and 7.8X10 9 /L) leukocyte-enriched PRP.
The high platelet concentration (4.8 times: the platelet and leukocyte amounts of the sample before centrifugation were respectively 10X 10) was obtained in the separation chamber B 9 L and 4.6X10 9 After centrifugation, the amounts of platelets and leukocytes in the sample were 159X 10, respectively 9 L and 1.6X10 9 L) lean leukocyte PRP.
Example 6
The aperture of the diatomite porous material in the centrifuge tube is 3 microns, and the capacity of the centrifuge tube is 7mL; after 3-4 mL of anticoagulated whole blood is placed in a separation chamber A, a centrifuge tube covering a tube orifice is placed in a centrifuge tube clamp and fixed on a horizontal centrifuge rotor platform, the included angle alpha between the axis Y of the centrifuge tube and the direction of a centrifugal force F is 0 degree (parallel), and the centrifuge tube is centrifuged for 3 minutes at 3000 Revolutions Per Minute (RPM).
The supernatant in separation chamber A was a high platelet concentration with red blood cells removed (1.8 times: the sample platelet and white blood cell amounts before centrifugation were 56X 10, respectively) 9 L and 4.5X10 9 The platelet and leukocyte amounts in the centrifuged samples were 98X 10, respectively 9 L and 4.4X10 9 /L)PRP。
The separation chamber B is depleted of white blood cells PRP (12 times the amount of platelets and white blood cells of the sample before centrifugation is 56×10 respectively) 9 L and 4.5X10 9 The platelet and leukocyte amounts in the centrifuged samples were 721X 10, respectively 9 L and 1.0X10 9 /L)。
Example 7
The aperture of the polycarbonate porous material in the centrifuge tube is 2 microns, and the capacity of the centrifuge tube is 7mL; after 3-4 mL of anticoagulated whole blood is placed in the separation chamber a, a centrifuge tube covering the tube orifice is placed in a centrifuge tube clamp and fixed on a horizontal centrifuge rotor platform, the included angle alpha=30 degrees between the axis Y of the centrifuge tube and the direction of the centrifugal force F, and the centrifuge tube is centrifuged for 5 minutes at 1500 Revolutions Per Minute (RPM).
The supernatant in separation chamber A was a high platelet concentration with red blood cells removed (2.8 times the amount of platelets and white blood cells of the sample before centrifugation, respectively, was X10) 9 L and 4.6X10 9 The platelet and leukocyte amounts in the centrifuged samples were 94X 10, respectively 9 L and 7.7X10 9 /L) leukocyte-enriched PRP.
The separation chamber B was depleted of PRP (4.1 times the amount of platelets and white blood cells in the sample before centrifugation was 33X 10, respectively) 9 L and 4.6X10 9 The platelet and leukocyte amounts in the centrifuged samples were 135X 10, respectively 9 L and 1.7X10 9 L) lean leukocyte PRP.
Example 8
The aperture of the polycarbonate porous material in the centrifuge tube is 5 microns, and the capacity of the centrifuge tube is 7mL; after 2-3 mL of anticoagulated whole blood is placed in a separation chamber A, a centrifuge tube covering a tube orifice is placed in a centrifuge tube clamp and fixed on a horizontal centrifuge rotor platform, and the included angle alpha=30 DEG between the central axis direction Y of the centrifuge tube and the direction of the centrifugal force F is separated for 5 minutes at 3000 Revolutions Per Minute (RPM).
Because the porous material has larger pore diameter and larger centrifugal speed (larger centrifugal force), most of the sample reaches the separation chamber B through the porous material to obtain the white blood cell-depleted PRP (1.7 times of the raw blood; the platelet and white blood cell amount of the sample before centrifugation are respectively 56 multiplied by 10) 9 L and 4.5X10 9 The platelet and leukocyte amounts in the centrifuged samples were 96X 10, respectively 9 L and 0.2X10 9 /L)。
Example 9
The aperture of the polycarbonate porous material in the centrifuge tube is 3 microns, and the capacity of the centrifuge tube is 7mL; after 2-3 mL of anticoagulated whole blood is placed in the separation chamber a, a centrifuge tube covering the tube orifice is placed in a centrifuge tube clamp and fixed on a horizontal centrifuge rotor platform, the included angle alpha=45 degrees between the axis Y of the centrifuge tube and the direction of the centrifugal force F, and the centrifuge tube is centrifuged for 5 minutes at 2000 Revolutions Per Minute (RPM).
PRP was not obtained in separation chamber A (sample platelet and leukocyte amounts before centrifugation were 85X 10, respectively) 9 L and 5.6X10 9 The platelet and leukocyte amounts in the centrifuged samples were 55X 10, respectively 9 L and 0.7X10 9 /L)。
The separation chamber B was subjected to ultra-high platelet concentration (7.6 times the amount of platelets and white blood cells in the sample before centrifugation was 85X 10, respectively) 9 L and 5.6X10 9 The platelet and leukocyte amounts in the centrifuged samples were 187X 10, respectively 9 L and 1.1X10 9 L) lean leukocyte PRP.
Example 10
The pore diameter of the porous material in the centrifuge tube is 3 micrometers, the capacity of the centrifuge tube is 7mL, and the porous material is prepared by polycarbonate track etching; after 2-3 mL of anticoagulated whole blood is placed in a separation chamber A, a centrifuge tube covering a tube orifice is placed in a centrifuge tube clamp and fixed on a horizontal centrifuge rotor platform, the included angle alpha=60 DEG between the central axis direction Y of the centrifuge tube and the direction of the centrifugal force F, and the centrifuge tube is centrifuged for 5 minutes at 3000 Revolutions Per Minute (RPM).
In the separation chamber ASupernatant was not PRP (platelet and leukocyte amounts of samples before centrifugation were 88X 10, respectively) 9 L and 2.8X10 9 The platelet and leukocyte amounts in the centrifuged samples were 59X 10, respectively 9 L and 0.3X10 9 /L)。
Obtained in separation chamber B was a high platelet concentration (3.3 times: the sample platelet and leukocyte amounts before centrifugation were 88X 10, respectively) 9 L and 2.8X10 9 The platelet and leukocyte amounts in the centrifuged samples were 291X 10, respectively 9 L and 1.4X10 9 L) lean leukocyte PRP.
Comparative example 1
As shown in fig. 2, in the conventional fixed angle rotor centrifugal apparatus, the center axis of the centrifugal tube has an angle θ with the centrifugal rotation axis of the centrifugal apparatus. The included angle θ=45 degrees (i.e., fixed angle rotor centrifuge) employed in this comparative example.
The aperture of the polycarbonate porous material in the centrifuge tube is 2 microns, and the capacity of the centrifuge tube is 7mL; after 3-4 mL of anticoagulated whole blood is placed in a separation chamber A, a centrifuge tube is placed in a fixed angle rotor centrifuge, and the whole blood is centrifuged for 1 minute at 3000 Revolutions Per Minute (RPM), at this time, the blood clings to the upper surface of the porous material due to gravity, and an additional extrusion force is applied to the porous material, so that the pore diameter of the porous material is increased or the porous material is locally ruptured.
It was found that PRP could not be prepared by centrifugation of blood for 1 min, i.e. all through the polycarbonate porous material into the separation chamber B.
Comparative example 2
Placing 2-3 mL of anticoagulated whole blood at the bottom of 7mL centrifuge tube, centrifuging at 3500 Revolutions Per Minute (RPM) for 5min by adopting the horizontal centrifuge rotor of FIG. 1, wherein the included angle between the axis Y of the centrifuge tube and the direction of centrifugal force F is alpha=45 DEG, and separating the whole blood (red blood cells at the bottom of the centrifuge tube and blood plasma at the upper part of the centrifuge tube) while the concentration of blood platelets in the blood plasma is not concentrated (the sample blood platelets and white blood cell amounts before centrifugation are 89×10 respectively) 9 L and 9.1X10 9 The platelet and leukocyte amounts in the centrifuged samples were 42X 10, respectively 9 L and 0.9X10 9 L), no acceptable PRP can be obtained. It can be seen that without porous material, horizontal centrifugation does not produce acceptable PRP.
Comparative example 3
Placing 2-3 mL of anticoagulated whole blood at the bottom of 7mL centrifuge tube, centrifuging at 1500 RPM for 2-5 min by adopting horizontal centrifuge rotor of figure 1, wherein the included angle alpha between the central axis direction Y of centrifuge tube and the direction of centrifugal force F is 0 degree (parallel), and separating whole blood (red blood cells at the bottom of centrifuge tube and blood plasma at the upper part of centrifuge tube) to obtain blood platelet concentration equivalent to that of original whole blood (sample blood platelet and white blood cell amount before centrifugation are 89×10 respectively) 9 L and 9.1X10 9 After centrifugation, the amounts of platelets and leukocytes in the sample were 127X 10, respectively 9 L and 12.4X10 9 /L), no acceptable platelet concentration of PRP could be produced (commercial PRP minimum platelet concentration 1.7 times that of raw blood, i.e., the RegenKit product of Table 1). It can be seen that without porous material, horizontal centrifugation does not produce acceptable PRP.
Claims (11)
1. A horizontal centrifugal rotor, comprising: a horizontal centrifugal rotor platform and a centrifugal tube clamp;
the horizontal centrifugal rotor platform is provided with a rotation central axis X, and a centrifugal pipe clamp is arranged on the horizontal centrifugal rotor platform;
the centrifugal tube clamp is used for placing and limiting a centrifugal tube, and keeps synchronous rotation with the horizontal centrifugal rotor platform, and the centrifugal tube is placed behind the centrifugal tube clamp and provided with a horizontal centrifugal tube axis Y;
the axis Y of the centrifugal tube and the rotation central axis X are always vertical in the start-stop stage of the horizontal centrifugal rotor platform; the centrifugal tube axis Y and the centrifugal force direction F have an included angle alpha, and the alpha angle is 0-60 degrees.
2. The horizontal centrifugal rotor according to claim 1, wherein a centrifugal pipe clamp positioning device and a centrifugal pipe clamp nest are fixedly connected to the horizontal centrifugal rotor platform;
the centrifuge tube clip is placed within the centrifuge tube clip nest, and the centrifuge tube clip positioning device positions the centrifuge tube clip at a desired location within the centrifuge tube clip nest.
3. A horizontal centrifugal rotor according to claim 2, wherein a plurality of said centrifugal tube clamp positioning means are provided, uniformly arranged on a horizontal centrifugal rotor platform around said rotation central axis X; the centrifugal pipe clamp nest quantity is the same with the centrifugal pipe clamp positioner quantity, evenly sets up on horizontal centrifugal rotor platform.
4. A horizontal centrifuge rotor according to claim 2 or 3, wherein the centrifuge tube clamps are provided with concentrically arranged positioning holes, and the centrifuge tube clamp positioning means has positioning protrusions cooperating with said positioning holes, whereby the angle α between the centrifuge tube axis Y and the direction of centrifugal force F is adjustable by cooperation of the positioning protrusions with different positioning holes on adjacent centrifuge tube clamps.
5. The horizontal centrifugal rotor according to claim 4, wherein the centrifugal pipe clamp positioning device is fixedly connected to one end of the horizontal centrifugal rotor platform far away from the rotation central axis X and is provided with a fixed point and a free end; the positioning protrusion is positioned at the free end, and the free end can do 90-degree reciprocating rotation around the fixed point towards the direction of the centrifugal pipe clamp.
6. A centrifugal device comprising a horizontal centrifugal rotor according to any one of claims 1-5.
7. A cell separation device, characterized in that: comprising the centrifuge apparatus and centrifuge tube of claim 6; the centrifuge tube comprises a separation chamber A and a separation chamber B, wherein the separation chamber A and the separation chamber B are separated by a porous material, and the separation chamber B is positioned at one end of the bottom of the centrifuge tube.
8. The cell separation apparatus of claim 7, wherein: the pore diameter of the porous material is 1-5 microns; the porous material is a high polymer material or an inorganic material.
9. The cell separation apparatus of claim 8, wherein: the high polymer material is selected from polycarbonate, polyester or polysulfone; the inorganic material is selected from diatomaceous earth.
10. The cell separation apparatus of claim 7, wherein: the porous material is fixed in a position of the separation chamber A near the bottom, and the separation chamber A is connected with the separation chamber B through threads.
11. A method of blood cell separation by a cell separation device according to any one of claims 7 to 10, comprising the steps of: and placing the anticoagulated whole blood in a separation chamber A, placing the centrifuge tube in a centrifuge tube clamp, wherein an included angle alpha is formed between the axis Y of the centrifuge tube and the direction of the centrifugal force F, and the rotation speed is Zhong Lixin-5 minutes every minute of 1500-4000.
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