WO2022252910A1

WO2022252910A1 - Piston-type biological liquid separation bottle having inner core and separation method thereof

Info

Publication number: WO2022252910A1
Application number: PCT/CN2022/091109
Authority: WO
Inventors: 魏东兵
Original assignee: 衷博瑞佳生物技术(上海)有限公司
Priority date: 2021-05-31
Filing date: 2022-05-06
Publication date: 2022-12-08
Also published as: CN113318287A

Abstract

A piston-type biological liquid separation bottle having an inner core and a separation method. The separation bottle comprises a liquid inlet, an inner core, a separation cavity, and a liquid outlet. The liquid inlet is separated from the liquid outlet; a biological liquid enters the separation cavity by means of the liquid inlet and the inner core; and after the biological liquid is separated in the separation cavity, part of the liquid is discharged by means of the liquid outlet, and the remaining liquid is discharged by means of the inner core and the liquid inlet. The method enables high-efficiency continuous processing of the biological liquid, without limiting the amount of liquid processed in a single time. The centrifugation path is long; the centrifugation time is sufficient; the centrifugation effect is better, and the cell recovery rate is higher; the inlet and outlet are separated; and the liquid inlet, separation cavity, flow guide cavity, liquid outlet, etc. can be cleaned, thus preventing the loss of target cells.

Description

Piston-type biological liquid separation cup with inner core and separation method thereof

technical field

The invention belongs to the field of biological liquid separation, and in particular relates to a piston-type biological liquid separation cup with an inner core and a separation method thereof.

Background technique

In modern biotechnology, it is often necessary to process biological fluids to obtain one or several target cells. Such as removing excess plasma and red blood cells in peripheral blood and umbilical cord blood to obtain stem cells or immune cells; or concentrating and washing the cultured cell liquid to remove excess waste liquid to obtain the target concentration of cell liquid. Since the volume of biological fluid is generally not fixed, for example, the volume of umbilical cord blood that needs to be processed is generally between 40ml-150ml, and the volume of cell fluid that needs to be concentrated and washed is generally between 200ml-50L. The above volume is not fixed. The current popular way to deal with biological liquids is to use a "piston type" separation cup with variable volume. For example, the patent No. CN1331610A discloses a system for separating biological fluid components, in which the supporting "piston type" separation cup is emphatically described. Its separation cup structure includes a hollow centrifugal treatment chamber, which has an axial biological fluid inlet/outlet; the treatment chamber has a movable piston, and under the action of the "air pump" of its supporting equipment, "suck" a certain amount of biological fluid into the treatment chamber. After processing, the piston moves upwards and squeezes out the processed biological fluid components through the outlet; the position of the piston is monitored through the optical device of the supporting equipment, and the pressure regulating valve device on the equipment is selectively connected to the processing chamber and the container, or Cut off their connections. Patent No. CN109294899A discloses "a method for preparing PBMC cells", which also includes a "piston centrifuge", which cooperates with the pneumatic device on the equipment to suck/exhaust biological liquid. Its structure is the same as that disclosed in CN1331610A. The structure of the cup is basically the same.

Piston-type separation cups can flexibly handle biological liquids with variable volumes, but the separation cups of the current technology have the following problems:

(1) The liquid volume for a single treatment is small, and the biological liquid cannot be processed continuously. Since the single treatment volume of the "piston type" separation cup cannot exceed its maximum volume (generally 220ml-250ml), when the treatment volume of biological liquid exceeds its maximum volume, multiple cycles must be carried out; if its maximum volume is 250ml, When the amount of treatment liquid is between 40ml-150ml, the treatment can be completed directly in one time (one cycle includes inhalation→centrifugation→exhaust waste liquid→collection of target cells); and when the treatment liquid volume is 2000ml of cell fluid, then at least 8 cycles are required, and the efficiency is very low;

(2) The target cells near the entrance are wasted, which reduces the target cell recovery rate. Because the inlet and the outlet of the separation cup are the same, every time the biological liquid to be separated is inhaled, some will inevitably remain at the inlet. After the centrifugation is completed, the "waste liquid" will be removed first, and the biological fluid containing the target cells at the inlet is also discharged together, wasting precious target cells. The more cycles, the more target cells are wasted.

(3) The closer the inhaled biological fluid is to the center of the rotation axis in each cycle, the shorter the centrifugation time (relative to the start of inhaling the biological fluid) and the less sufficient the centrifugation. Whether the centrifugation of the biological liquid is sufficient or not is closely related to the centrifugation time and the centrifugal force; due to the centrifugal force G=ω ² r (where ω is the rotational speed and r is the centrifugal radius), at the same rotational speed, the part of the liquid that is closer to the axis of the top of the separation cup The shorter the centrifugal radius, the smaller the centrifugal force and the less sufficient the centrifugal force. At the end of centrifugation, a small number of target cells that have not had enough time to centrifuge will be mixed in the waste liquid and discharged together, resulting in a low recovery rate of target cells.

Contents of the invention

In order to solve at least one of the above-mentioned technical problems, the technical solution adopted in the present invention is to provide a piston-type biological liquid separation cup with an inner core and its separation method, which can continuously process biological liquid with high efficiency, and the amount of single-treatment liquid is not limited. ; It can avoid the loss of target cells; the centrifugation path is long, the centrifugation time is sufficient, the centrifugation effect is good, and the cell recovery rate is higher.

In order to achieve at least one of the above-mentioned purposes, the technical solution adopted in the present invention is:

The invention provides a piston-type biological liquid separation cup with an inner core, the separation cup includes a liquid inlet, an inner core, a separation chamber and a liquid outlet; the liquid inlet is set separately from the liquid outlet; the biological liquid passes through the The liquid inlet and the inner core enter the separation chamber; after the biological liquid is separated in the separation chamber, part of the liquid is discharged through the liquid outlet, and the rest of the liquid is discharged through the inner core and the liquid inlet.

Further, the inner core is a hollow cylindrical body of revolution, and the inner core includes a hemispherical converging hole, a diversion cone, an annular baffle, a waist-shaped hole and a strip-shaped hole; the biological liquid passes through the hemispherical The hemispherical concentrating hole, diversion cone, circular baffle and waist-shaped hole enter the separation chamber, and the hemispherical converging hole, diversion cone, circular baffle and waist-shaped hole form a diversion channel; said strip-shaped hole fluidly communicates said separation chamber with a liquid outlet.

Further, it also includes a cup and a piston; the cup is a hollow transparent cylinder, the inner core is fixed in the cup, the piston is slidably arranged between the inner core and the cup, and is connected with the The inner core and the cup are in sliding and sealing fit; the cup, the inner core and the piston are coaxially arranged, and the cavity between the inner surface of the cup, the drainage cone above the piston and the outer surface of the inner core forms the the separation chamber.

Further, the outer surface of the bottom of the cup body is provided with a first flange and a second flange, and the end surface of the bottom is provided with a stepped end surface; it also includes a base, the base is disc-shaped, and the base of the base is The bottom is provided with an air inlet, the upper part of the base is provided with a limit ring, a limit groove and a third flange, and a plurality of small holes for ventilation are evenly distributed on the limit ring, and the small holes will The air inlet hole is in fluid communication with the air pressure chamber below the piston; a hollow annular bottom cover is provided outside the base, and a plurality of evenly distributed buckles are arranged on the edge of the bottom cover, and the buckles are connected with the The second flange cooperates to fix the base on the bottom of the cup body.

Further, it also includes a fixed head, the fixed head is a hollow multi-stage stepped columnar body, the fixed head includes a first stepped shaft, a second stepped shaft and a circular disk; the liquid inlet is provided through the first On a stepped shaft, the liquid outlet is provided through on the second stepped shaft; the bottom of the circular disk is provided with a first protruding ring, a second protruding ring and a third protruding ring, and on the first protruding ring An outer flange, a first groove and a second groove are sequentially arranged between the ring, the second protruding ring and the third protruding ring from outside to inside.

Further, it also includes a first inner liner and a second inner liner, both of which are in the shape of an inverted funnel; the first inner liner includes a first hollow cylinder with a small end and a first large end The end cone body, the outside of the first small-end hollow cylinder is provided with a stepped shaft and an annular groove; the inner surface of the first small-end hollow cylinder is provided with a plurality of first support bars evenly distributed along the axial direction; The second lining includes a second small-end hollow cylinder and a second large-end cone; a hollow stepped hole is provided inside the second small-end hollow cylinder; A plurality of second support strips evenly distributed; a second dynamic sealing ring is arranged on the annular groove of the first inner liner; the stepped shaft of the first small-end hollow cylinder on the first inner liner is inserted in a shape-fitting manner In the second stepped shaft of the fixed head, it is abutted and fixed with the third protruding ring; after the second small-end hollow cylinder of the second lining passes through the first small-end hollow cylinder, the end The stepped shaft of the fixed head is bonded to the stepped hole of the fixed head, the outer surface of the hollow cylinder at the second small end abuts against the first support bar inside the hollow cylinder with the first lining, and the second inner The second support strip of the lining abuts against the inner side of the first large-end cone of the first lining; it also includes a guide tube, and the guide tube is bonded in the hollow stepped hole; the guide tube The inner surface of the pipe, the first small end hollow cylinder and the liquid inlet form the liquid inlet channel; the strip hole, the outer surface of the second large end cone, the inner surface of the first small end hollow cylinder, the second small end hollow The outer side of the cylinder, the inner side of the hollow cylinder at the first small end and the liquid outlet form a liquid outlet channel.

Further, it also includes a bearing seat, the bearing seat is a cylindrical hollow rotating body, the upper part of the bearing seat includes an upper convex ring, a third groove, and a through hole is arranged in the middle, and the upper convex ring and the third groove are arranged in the middle. There is an upper step ring surface between the through holes; the lower part of the bearing seat is provided with an annular step surface; a first static sealing ring is arranged in the third groove, and a first static sealing ring is arranged on the upper step ring surface dynamic sealing ring; the upper protruding ring is bonded to the first groove, and the first protruding ring is bonded to the third groove; the first protruding ring and the second protruding ring are respectively bonded to the The first static sealing ring abuts against the first dynamic sealing ring.

Further, it also includes a cup cover, which includes a third small-end hollow cylinder and a third large-end cone, and a fourth convex ring and a fourth groove are arranged on the bottom surface of the third large-end cone ; The upper end of the cup is provided with a fifth protruding ring and a fifth groove; the fourth protruding ring is bonded in the fifth groove, and the fifth protruding ring is bonded in the fourth groove in the slot.

Further, it also includes a bearing, the bearing is installed in the bearing hole of the bearing seat through the interference fit of the outer ring; the hollow cylinder at the third small end of the cup cover is interference fit and positioned with the inner ring of the bearing , inserted into the second groove; and the outer surface of the third small-end hollow cylinder is in interference fit with the first dynamic sealing ring, and there is a rotary seal between the two; the third small-end hollow cylinder The inner surface is in interference fit with the second dynamic sealing ring, and the two are rotated and sealed.

The present invention also provides a separation method of the piston-type biological liquid separation cup with an inner core according to the claims, the separation method includes single treatment and continuous treatment;

The steps of the single treatment are:

a The centrifuge on the separation equipment drives the separation cup to rotate at high speed;

bUsing the peristaltic pump as the power, a certain amount of gradient liquid ficoll is transported to the hemispherical converging hole at the bottom of the inner core through the liquid inlet channel; under the action of centrifugal force, the gradient liquid climbs rapidly along the diversion channel to the annular stopper Plate, and then quickly climb into the separation chamber along the waist hole and drainage cone;

c Use a peristaltic pump to transport the biological fluid to be separated from the liquid inlet channel to the hemispherical gathering hole at the bottom of the inner core; the biological fluid will quickly enter the separation chamber along the diversion channel; when the biological fluid completely enters the separation cup, all the biological fluid The liquid will be located in the separation chamber; after a period of centrifugation, the heavier red blood cells will be at the outermost side of the separation chamber, and then the gradient liquid, buffy coat and plasma will be inward sequentially;

d Use the air pump to push the piston, so that the volume of the separation chamber gradually decreases, and the inner plasma and buffy coat are pushed out in turn; cooperate with the valve, color sensor, pipeline component sensor, component collection bag and waste liquid on the separation device bag to collect the required ingredients;

e When only red blood cells and gradient liquid are left in the separation chamber, the air pump pumps air in reverse, the actuating piston moves downward, and at the same time the centrifuge stops slowly; the liquid in the separation chamber will flow into the hemispherical concentrating hole under the action of gravity Under the action of the peristaltic pump, the remaining liquid is sucked away from the guide tube to complete the separation;

The steps of the continuous processing are:

The centrifuge on the A separation device drives the separation cup to rotate at high speed;

The B equipment is powered by a peristaltic pump to transport the biological fluid from the liquid inlet channel to the hemispherical flow collecting hole at the bottom of the inner core; under the action of centrifugal force, the biological fluid climbs rapidly along the diversion channel to the circular baffle, Then climb rapidly along the waist hole and the drainage cone to the separation cavity, and the cell fluid gradually fills the separation cavity; during the process of filling the separation cavity with the biological fluid, the cells in the biological fluid are distributed in the separation cavity under the action of centrifugal force. The outermost chamber, while the waste liquid is at the innermost side of the separation chamber;

C With the continuous input of biological fluid, the innermost waste liquid without cells in the separation chamber is squeezed out; when the biological liquid is completely input into the separation cup, the outer side of the separation chamber is cells, the inner side is waste liquid, and the sum of the volumes is the separation volume. The maximum dynamic volume of the cup;

DStart the air pump, push the piston, cooperate with the gravity sensor, optical sensor, valve and other components on the separation equipment, until there is a certain volume in the separation cup, stop pushing the piston;

E The centrifuge is stopped, and the air pump is started, and the air is pumped in reverse, so that the piston moves to the bottom; under the action of gravity, all the biological liquid in the separation cup is collected at the hemispherical flow hole at the bottom of the inner core; then the peristaltic pump is started , absorb the remaining biological fluid from the guide tube;

F suction a small amount of cleaning solution from the liquid outlet, and start the centrifuge to clean the liquid outlet, liquid outlet channel, separation chamber and diversion channel; then start the peristaltic pump again to suck the residual cells from the liquid inlet channel into the product bag.

Compared with the prior art, the invention provides a piston-type biological liquid separation cup with an inner core and its separation method. Compared with the prior art, the invention has the beneficial effects of:

(1) Compared with ordinary piston-type separation cups, by increasing the inner core, a fluid channel from the inlet to the outlet is established in the separation cup, which can efficiently and continuously process biological liquids, and the amount of single-treatment liquids is not limited; it can Separation of small volumes of biological fluids such as umbilical cord blood, peripheral blood, and bone marrow, as well as concentration and washing of large volumes of cell fluids;

(2) By increasing the inner core, the minimum centrifugal force of the biological liquid in the separation chamber is limited, a better centrifugal effect is obtained, and the recovery rate of the target cells will be higher;

(3) Establish a fluid channel for the biological liquid to flow from the inlet of the separation cup, the central guide tube, and the guide chamber to the bottom of the separation chamber, and then diffuse upward to the liquid outlet; from the inlet to the outlet, the centrifugation path is long and the centrifugation time is sufficient , the centrifugation effect is better, and the cell recovery rate is higher;

(4) The inlet and outlet of the separation cup are separated, which can clean the liquid inlet, separation chamber, diversion chamber, liquid outlet, etc., avoiding the loss of target cells and improving the recovery rate of cells.

Description of drawings

Fig. 1 is the structural representation of the piston type biological liquid separation cup with inner core of the present invention;

Fig. 2 is the explosion schematic diagram of the piston-type biological liquid separation cup with inner core of the present invention;

Fig. 3 is the structural representation of fixed head of the present invention;

Fig. 4 is the structural representation of the first liner of the present invention;

Fig. 5 is a schematic diagram of a section along B-B in Fig. 4;

Fig. 6 is the structural representation of the second liner of the present invention;

Fig. 7 is the view of M direction in Fig. 6;

Fig. 8 is a structural schematic diagram of the bearing seat of the present invention;

Fig. 9 is a schematic structural view of the cup lid of the present invention;

Fig. 10 is a schematic structural view of the cup body of the present invention;

Fig. 11 is a schematic structural view of the piston of the present invention;

Fig. 12 is a schematic structural diagram of the kernel of the present invention;

Fig. 13 is a schematic structural view of the base of the present invention;

Fig. 14 is a schematic diagram of the internal structure of the base of the present invention;

15-19 are process schematic diagrams of the separation cup of the present invention when the volume of separated biological fluid can be processed in a single time;

20-23 are schematic diagrams of the separation cup of the present invention when the volume of separated biological fluid needs to be processed continuously.

Among them, 1 fixed head, 1A first stepped shaft, 1A1 stepped hole, 1B second stepped shaft, 1C circular disc, 1C1 first convex ring, 1C2 second convex ring, 1C3 third convex ring, 1C4 outer flange, 1C5 first groove, 1C6 second groove, 1D liquid inlet, liquid outlet 1E, 2 first static seal ring, 3 first dynamic seal ring, 4 bearing seat, 4A upper convex ring, 4B third groove, 4C Through hole, 4D upper stepped annular surface, 4E annular stepped surface, 5 bearings, 6 cup cover, 6A third small end hollow cylinder, 6B third large end cone, 6B1 fourth convex ring, 6B2 fourth groove, 7 The second dynamic sealing ring, 8 the first inner lining, 8A the first small end hollow cylinder, 8A1 stepped shaft, 8A2 annular groove, 8B the first large end cone, 8C the first support bar, 9 the second inner lining, 9A The second small end hollow cylinder, 9A1 hollow step hole, 9B second large end cone, 9B1 second support bar, 10 diversion tube, 11 inner core, 11A first ladder column, 11B second ladder column, 11C third ladder Column, 11D step end face, 11E strip hole, 11F waist hole, 11G circular baffle, 11H diversion cone surface, 11J hemispherical converging hole, 12 cup body, 12A fifth convex ring, 12B fifth groove Groove, 12C first flange, 12D second flange, 12E stepped end face, 13 third piston sealing ring, 14 first piston sealing ring, 15 piston, 15A sixth groove, 15B seventh groove, 15C eighth Groove, 15D ninth groove, 15E drainage cone, 15F air pressure chamber, 16 second piston sealing ring, 17 second static sealing ring, 18 base, 18A air inlet, 18B limit ring, 18C limit groove, 18D third flange, 18E small hole, 19 bottom cover.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific examples. Please note that the embodiments described below are exemplary only for explaining the present invention, and should not be construed as limiting the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification.

In the description of the present invention, it should be understood that the terms "first" and "second" are used for description purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Thus, a feature defined as "first" and "second" may explicitly or implicitly include one or more of these features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

In the present invention, unless otherwise clearly specified and limited, terms such as "installation", "connection", "connection" and "fixation" should be understood in a broad sense, for example, it can be a fixed connection or a detachable connection , or integrated; it can be mechanically connected or electrically connected; it can be directly connected or indirectly connected through an intermediary, and it can be the internal communication of two components or the interaction relationship between two components. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present invention according to specific situations.

Below, the piston-type biological liquid separation cup with an inner core and its separation method provided by the present invention will be described in detail through specific examples:

As shown in the accompanying drawings 1-14, the piston-type biological liquid separation cup with inner core provided by the present invention mainly includes a fixed head 1, a first static seal ring 2, a first dynamic seal ring 3, a bearing seat 4, a bearing 5, a cup Cover 6, second dynamic sealing ring 7, first inner lining 8, second inner lining 9, draft tube 10, inner core 11, cup body 12, third piston sealing ring 13, first piston sealing ring 14, piston 15 , The second piston seal ring 16, the second static seal ring 17, the base 18 and the bottom cover 19 and other parts.

As shown in FIG. 3 , the main body of the fixing head 1 is a hollow multi-level stepped columnar body, including a first stepped shaft 1A, a second stepped shaft 1B, and a circular disc 1C. The first stepped shaft 1A is provided with a hollow columnar liquid inlet 1D, and the first stepped shaft 1A is also provided with a stepped hole 1A1; the second stepped shaft 1B is provided with a hollow columnar liquid outlet 1E; the bottom of the circular disc 1C A first protruding ring 1C1 , a second protruding ring 1C2 and a third protruding ring 1C3 are provided, and an outer flange 1C4 , a first groove 1C5 and a second groove 1C6 are sequentially arranged between the above protruding rings from outside to inside.

As shown in FIGS. 4-5 , the first inner liner 8 is shaped like an inverted funnel, and includes a first small-end hollow cylinder 8A and a first large-end cone 8B. A stepped shaft 8A1 and an annular groove 8A2 are provided on the outside of the first small-end hollow cylinder 8A; three first support bars 8C evenly distributed along the axial direction are provided on the inner surface of the first small-end hollow cylinder 8A.

As shown in Figures 6-7, the second lining 9 is also shaped like an inverted funnel, including a hollow cylinder 9A at the second small end and a cone 9B at the second large end. A hollow stepped hole 9A1 is provided inside the second small-end hollow cylinder 9A; three second support bars 9B1 are evenly distributed on the outer surface of the second large-end cone 9B.

As shown in Figure 8, the bearing seat 4 is a cylindrical hollow rotating body, the upper part of the bearing seat 4 includes an upper convex ring 4A, a third groove 4B, and a through hole 4C is arranged in the middle, and the upper convex ring 4A and the through hole There is an upper stepped annular surface 4D between 4C; the lower part of the bearing seat 4 is provided with an annular stepped surface 4E.

The second dynamic sealing ring 7 is arranged on the annular groove 8A2 of the first inner liner 8; the stepped shaft 8A1 of the first small-end hollow cylinder 8A on the first inner liner 8 is inserted into the second step of the fixed head 1 in a shape fit In the shaft 1B, it is abutted and fixed with the third protruding ring 1C3. After the second small-end hollow cylinder 9A of the second lining 9 passes through the first small-end hollow cylinder 8A, the stepped shaft at the end is bonded to the stepped hole 1A1 of the fixed head 1, and the outer surface of the second small-end hollow cylinder 9A The surface abuts against the three first support strips 8C inside the hollow cylinder 8A of the first lining, while the three second support strips 9B1 of the second lining 9 are in contact with the first large-end cone 8B of the first lining 8 inner contact. The guide tube 10 is a hollow long round tube, and the outer wall and upper end surface of the guide tube 10 are bonded to the hollow stepped hole 9A1 in the second lining 9 . The bearing 5 is installed in the bearing hole of the bearing seat 4 through the interference fit of the outer ring of the bearing; the first static sealing ring 2 is arranged in the third groove 4B of the bearing seat 4, and the first dynamic sealing ring 3 is arranged in the Go up the stairs on Torus 4D. The upper convex ring 4A of the bearing seat 4 is bonded to the first groove 1C5 of the fixed head 1 , and the first convex ring 1C1 of the fixed head 1 is bonded to the third groove 4B of the bearing seat 4 . The first protruding ring 1C1 and the second protruding ring 1C2 of the fixed head 1 are squeezed into contact with the first static seal ring 2 and the first dynamic seal ring 3 installed on the bearing seat 4 respectively, and the joint between the fixed head 1 and the bearing seat 4 seal between. To sum up, the fixed head 1, the first inner liner 8, the second inner liner 9, the guide tube 10 and the bearing seat 4 are fixed together to form a stationary component of the separation cup, that is, it is in a static state when the separation cup is separated.

As shown in Figure 9, the cup cover 6 is a similar inverted funnel-shaped body of revolution, including a third small-end hollow cylinder 6A and a third large-end cone 6B, and a fourth protrusion is arranged on the bottom end surface of the third large-end cone 6B. ring 6B1 and fourth groove 6B2.

As shown in FIG. 10 , the cup body 12 is a hollow transparent cylinder. The upper end of the cup body 12 is provided with a fifth protruding ring 12A and a fifth groove 12B matching the cup cover, and a stepped cylindrical surface is provided inside the upper end. A first flange 12C and a second flange 12D are provided on the outer surface of the bottom of the cup body 12 , and a stepped end surface 12E is provided on the end surface of the bottom.

As shown in FIG. 11 , the piston 15 is a hollow cylindrical rotating body. The outer side of the piston 15 is provided with the sixth groove 15A and the seventh groove 15B for installing the sealing ring; the inner side of the piston 15 is provided with the eighth groove 15C and the ninth groove 15D for installing the sealing ring; There is a conical surface 15E for drainage; the bottom of the piston 15 is provided with an air pressure chamber 15F.

As shown in FIG. 12 , the inner core 11 is a stepped cylindrical shell. The inner core 11 includes a first stepped column 11A, a second stepped column 11B and a third stepped column 11C; there is a stepped end surface 11D between the first stepped column 11A and the second stepped column 11B, and the second stepped column 11B and the third stepped column 11C There are four strip-shaped holes 11E evenly distributed at the junction of the third stepped column 11C, and evenly distributed waist-shaped holes 11F are arranged on the third stepped column 11C. The inner side of the third stepped column 11C is provided with an annular baffle 11G, a diversion cone Surface 11H and hemispherical focus hole 11J.

As shown in Figures 13-14, the base 18 is disc-shaped. The bottom of the base 18 is provided with an air inlet 18A, and the upper part of the base 18 is provided with a limiting ring 18B, a limiting groove 18C and a third flange 18D, and four holes 18E for ventilation are evenly distributed on the limiting ring 18B.

The first stepped column 11A, the second stepped column 11B and the stepped end surface 11D of the inner core 11 are bonded to the stepped cylindrical surface of the cup body 12 and positioned. The first piston sealing ring 14 and the second piston sealing ring 16 are respectively installed in the sixth groove 15A and the seventh groove 15B of the piston 15; Three sealing rings 13. The piston 15 is slidably arranged on the inner core 11, and can slide between the inner core 11 and the cup body 12, and the first piston sealing ring 14 and the second piston sealing ring 16 contact and seal with the inner surface of the cup body 12, and the third sealing ring Ring 13 is in contact with and seals against the outer surface of inner core 11 . The base 18 is arranged on the bottom of the cup body 12, wherein a second static sealing ring 17 is arranged on the stepped end face 12E of the bottom of the cup body 12, and the third flange 18D of the base 18 is in contact with the end face of the cup body 12, and the second The static sealing ring 17 is compressed; the bottom end surface of the inner core 11 is inserted into the limiting groove 18C of the base 18 . A hollow annular bottom cover 19 is arranged on the outside of the base 18, and a plurality of evenly distributed buckles are arranged on the edge of the bottom cover 19, and the buckles cooperate with the second flange 12D of the cup body 12 to fix the base 18 on the cup body 12 bottom. The cup cover 6 is bonded to the upper part of the cup body 12, wherein the fourth protruding ring 6B1 of the cup cover 6 is bonded in the fifth groove 12B of the cup body 12, and the fifth protruding ring 12A of the cup body 12 is bonded to the cup cover. 6 in the fourth trench 6B2. To sum up, the cup cover 6, the inner core 11, the cup body 12, the piston 15, the base 18 and the bottom cover 19 constitute the rotating assembly of the separation cup, that is, the separation cup is in a state of high-speed rotation when separating.

The third small-end hollow cylinder 6A of the cup cover 6 in the rotating assembly is interference fit and positioned with the inner ring of the bearing 5, and is inserted into the second groove 1C6 of the fixed head 1; and the outer surface of the third small-end hollow cylinder 6A The surface is in interference fit with the first dynamic sealing ring 3, and the two can be rotated and sealed; the inner surface of the third small-end hollow cylinder 6A is in interference fit with the second dynamic sealing ring 7, and the two can also be rotated and sealed; thereby realizing The rotating support sealing connection is carried out between the rotating component and the stationary component through the bearing.

In the separating cup provided by the present invention, the inner surface of the first small-end hollow cylinder 8A of the draft tube 10, the second lining 8, and the liquid inlet 1D at the top of the fixed head 1 form the liquid inlet channel; the hemispherical gathering of the inner core 11 Orifice 11J, diversion cone surface 11H, annular baffle plate 11G and waist-shaped hole 11F constitute a diversion channel; the inner surface of cup body 12, diversion cone surface 15E and the outer surface of inner core 11 form a separation chamber, When the piston 15 is at the bottom, the dynamic volume of the separation cup is the largest, preferably 250ml; when the piston 15 moves upwards, the separation chamber will gradually become smaller, and when the piston 15 moves to the top, the dynamic volume of the separation cup is the smallest, preferably 250ml 30ml; strip hole 11E above the inner core 11, the outer surface of the second large-end cone 9B, the inner surface of the first small-end hollow cylinder 8A, the outside of the second small-end hollow cylinder 9A and the first small-end hollow cylinder 8A The liquid outlet passage is formed between the inner side of the inner side and the liquid outlet 1E of the fixed head 1; the air inlet 18A of the base 18, the small hole 18E on the limit ring 18B, and the air pressure chamber 15F at the bottom of the piston 15 together form the air inlet and outlet passages. The air inlet and air outlet channels are not connected to the separation chamber at any time. In summary, when the separation cup provided by the present invention is separated, the biological fluid is separated through the liquid inlet channel, the flow guide channel, the separation chamber and the liquid outlet channel in sequence, so that a fluid channel from the liquid inlet to the liquid outlet can be established in the separation cup , the centrifugation path is long, the centrifugation time is sufficient, and the centrifugation effect is better; and the inlet and outlet are separated, which can clean the liquid inlet, separation chamber, diversion chamber, liquid outlet, etc., to avoid the loss of target cells; at the same time, through the inlet and outlet The gas outlet channel actuates the piston, which can change the volume of the separation chamber, and can process biological liquid continuously with high efficiency, and the amount of liquid processed at a time is not limited.

The first static sealing ring 2 and the second static sealing ring 17 provided by the present invention are preferably common rubber rings, preferably made of NBR; the first dynamic sealing ring 3 and the second dynamic sealing ring 7 are preferably fluorine rubber rings with smooth surfaces ; The first piston seal ring 14, the second piston seal ring 16 and the third piston seal ring 13 are preferably smooth silicone rings.

According to the separation method of the above-mentioned piston-type biological liquid separation cup with inner core provided by the present invention, the specific introduction is as follows:

1. As shown in attached drawings 15-19, when the volume of the biological fluid to be separated is between 40-250ml, specifically take the separation of 120ml of umbilical cord blood + 30ml of gradient liquid as an example, including the following steps:

a The centrifuge on the separation device drives the rotating assembly of the separation cup to rotate at high speed;

bUsing a peristaltic pump or other power source as the power, a certain amount of gradient liquid ficoll is transported to the hemispherical concentrating hole at the bottom of the inner core through the liquid inlet channel; under the action of centrifugal force, the gradient liquid climbs rapidly along the diversion channel to At the ring-shaped baffle, then quickly climb into the separation chamber along the waist hole and the drainage cone;

c Use a peristaltic pump to transport the cord blood to be separated from the liquid inlet channel to the hemispherical converging hole at the bottom of the inner core; similarly, the cord blood will quickly enter the separation chamber along the diversion channel; when the cord blood completely enters the separation cup, all All cord blood will be located in the separation chamber. After a period of centrifugation, the heavier red blood cells are at the outermost side of the separation chamber, and then the gradient solution, buffy coat and plasma are inward in sequence;

d Use an air pump to push the piston through the air intake and air outlet channels to gradually reduce the volume of the separation chamber, and push out the inner plasma and buffy coat in sequence; cooperate with the valve, color sensor, pipeline component sensor, Component collection bag and waste bag to collect the required components;

e When only red blood cells and gradient liquid are left in the separation chamber, the air pump pumps air in reverse, the actuating piston moves downward, and at the same time the centrifuge stops slowly; the liquid in the separation chamber will flow into the hemispherical concentrating hole under the action of gravity Under the action of the peristaltic pump, the remaining liquid is sucked away from the guide tube to complete the separation.

2. As shown in Figures 20-23, when the volume of the biological fluid to be separated is greater than 250ml, specifically, the cell fluid with a volume of 1000ml and a cell concentration of 1× ¹⁰⁷ cells/ml is concentrated to a volume of 100ml. For 1 x 10 ^cells /ml as an example, the following steps are included:

A The centrifuge on the separation equipment drives the rotating assembly of the separation cup to rotate at high speed;

Device B is driven by a peristaltic pump or other power source, and the cell fluid is transported from the liquid inlet channel to the hemispherical converging hole at the bottom of the inner core; under the action of centrifugal force, the cell fluid quickly climbs to the circular shape along the diversion channel Then climb up to the separation chamber quickly along the waist-shaped hole and the drainage cone surface, and the cell fluid gradually fills the separation chamber; when the cell fluid fills the separation chamber, the cells in the cell fluid are under the action of centrifugal force The bottom is distributed on the outermost side of the separation chamber, while the waste liquid is on the innermost side of the separation chamber;

C With the continuous input of cell fluid, the innermost cell-free waste liquid in the separation chamber is squeezed out; when the cell liquid is completely input into the separation cup, the outer side of the separation chamber is cells, the inner side is waste liquid, and the sum of the volume is separation The maximum dynamic volume of the cup is 250ml;

DStart the air pump, push the piston, cooperate with the gravity sensor, optical sensor, valve and other components on the separation equipment, until the remaining volume in the separation cup is 100ml, stop pushing the piston;

E The centrifuge stops, and the air pump is started, and the air is pumped in reverse, so that the piston moves to the bottom; under the action of gravity, all the cell fluid in the separation cup is collected at the hemispherical flow hole at the bottom of the inner core; then the peristaltic pump is started , absorb the remaining cell fluid from the guide tube;

F suction a small amount of cleaning solution from the liquid outlet, and start the centrifuge to clean the liquid outlet, liquid outlet channel, separation chamber and diversion channel; then start the peristaltic pump again to suck the residual cells from the liquid inlet channel into the product bag; complete continuous concentration biological fluids.

In the present invention, unless otherwise clearly specified and limited, the first feature may be in direct contact with the first feature or the first and second feature may be in direct contact with the second feature through an intermediary. touch. Moreover, "above", "above" and "above" the first feature on the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. "Below", "beneath" and "beneath" the first feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is less horizontally than the second feature.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.

Claims

A piston-type biological liquid separation cup with an inner core, characterized in that the separation cup includes a liquid inlet, an inner core, a separation chamber and a liquid outlet; the liquid inlet is separated from the liquid outlet; the biological liquid passes through the The liquid inlet and the inner core enter the separation chamber; after the biological liquid is separated in the separation chamber, part of the liquid is discharged through the liquid outlet, and the rest of the liquid is discharged through the inner core and the liquid inlet.
The piston-type biological liquid separation cup with an inner core according to claim 1, wherein the inner core is a hollow cylindrical body of revolution, and the inner core includes a hemispherical converging hole, a diversion cone, and an annular baffle , waist-shaped hole and strip-shaped hole; the biological liquid enters the separation chamber through the hemispherical converging hole, diversion cone, circular baffle and waist-shaped hole in turn, and the hemispherical converging hole , a diversion cone, a circular baffle and a waist-shaped hole to form a diversion channel; the strip-shaped hole fluidly communicates the separation chamber with the liquid outlet.
The piston-type biological liquid separation cup with an inner core according to claim 2, further comprising a cup body and a piston; the cup body is a hollow transparent cylinder, and the inner core is fixed in the cup body, so The piston is slidably arranged between the inner core and the cup body, and is in sliding and sealing cooperation with the inner core and the cup body; The cavity between the drainage cone surface and the outer surface of the inner core forms the separation cavity.
The piston-type biological liquid separation cup with an inner core according to claim 3, wherein a first flange and a second flange are provided on the outer surface of the bottom of the cup body, and a step is provided on the end surface of the bottom The end surface; also includes a base, the base is disc-shaped, the bottom of the base is provided with an air inlet, the upper part of the base is provided with a limiting ring, a limiting groove and a third flange, and the limiting ring is A plurality of small holes for ventilation are evenly distributed, and the small holes connect the air inlet hole with the air pressure chamber below the piston; a hollow annular bottom cover is provided outside the base, and the bottom The edge of the cover is provided with a plurality of evenly distributed buckles, and the buckles cooperate with the second flange to fix the base on the bottom of the cup body.
The piston-type biological liquid separation cup with an inner core according to claim 3, characterized in that it also includes a fixed head, the fixed head is a hollow multi-stage stepped columnar body, and the fixed head includes a first stepped shaft, a second Two stepped shafts and a circular disc; the liquid inlet is provided through the first stepped shaft, and the liquid outlet is provided through the second stepped shaft; the bottom of the circular disc is provided with a first protrusion Ring, second protruding ring and third protruding ring, between the first protruding ring, the second protruding ring and the third protruding ring, an outer flange, a first groove and a second groove are sequentially arranged from outside to inside groove.
The piston-type biological liquid separation cup with an inner core according to claim 5, further comprising a first inner liner and a second inner liner, both of the first inner liner and the second inner liner are shaped like an inverted funnel ; The first inner liner includes a first small-end hollow cylinder and a first large-end cone, and the outside of the first small-end hollow cylinder is provided with a stepped shaft and an annular groove; the first small-end hollow cylinder The inner surface is provided with a plurality of first support strips evenly distributed along the axial direction; the second lining includes a second small-end hollow cylinder and a second large-end cone; the inside of the second small-end hollow cylinder is provided with Hollow step hole; the outer surface of the second large-end cone is provided with a plurality of second support bars evenly distributed; the annular groove of the first lining is provided with a second dynamic sealing ring; the first The stepped shaft of the first small-end hollow cylinder on the inner liner is inserted into the second stepped shaft of the fixed head in a shape fit, and is abutted and fixed with the third protruding ring; the second inner liner of the second inner liner After the small-end hollow cylinder passes through the first small-end hollow cylinder, the stepped shaft at the end is bonded to the stepped hole of the fixed head, and the outer surface of the second small-end hollow cylinder is connected to the first inner surface. The first support bar lining the inside of the hollow cylinder abuts, and at the same time, the second support bar of the second inner liner abuts against the inner side of the first large-end cone of the first inner liner; a draft tube is also included, so The guide tube is bonded in the hollow step hole; the inner surface of the guide tube, the first small-end hollow cylinder and the liquid inlet form a liquid inlet channel; the strip-shaped hole, the second large-end cone The outer surface, the inner surface of the hollow cylinder at the first small end, the outer side of the hollow cylinder at the second small end, the inner side of the hollow cylinder at the first small end and the liquid outlet form a liquid outlet channel.
The piston-type biological liquid separation cup with inner core according to claim 6, is characterized in that it also includes a bearing seat, the bearing seat is a cylindrical hollow rotating body, and the upper part of the bearing seat includes an upper convex ring, The third groove is provided with a through hole in the middle, and an upper stepped ring surface is provided between the upper convex ring and the through hole; the lower part of the bearing seat is provided with an annular stepped surface; the third groove is provided with There is a first static seal ring, and a first dynamic seal ring is provided on the upper step ring surface; the upper protruding ring is bonded to the first groove, and the first protruding ring is bonded to the first In the three grooves: the first protruding ring and the second protruding ring abut against the first static sealing ring and the first dynamic sealing ring respectively.
The piston-type biological liquid separation cup with an inner core according to claim 7, further comprising a cup cover, the cup cover includes a hollow cylinder at a third small end and a cone at a third large end, and the third large end A fourth protruding ring and a fourth groove are provided on the bottom end surface of the end cone; a fifth protruding ring and a fifth groove are provided on the upper end of the cup; the fourth protruding ring is bonded on the fifth In the groove, the fifth protruding ring is bonded in the fourth groove.
The piston-type biological liquid separation cup with an inner core according to claim 8, is characterized in that it also includes a bearing, and the bearing is installed in the bearing hole of the bearing seat through the interference fit of the outer ring; The third hollow cylinder at the small end is interference fit and positioned with the inner ring of the bearing, and inserted into the second groove; and the outer surface of the hollow cylinder at the third small end interferes with the first dynamic sealing ring The inner surface of the hollow cylinder at the third small end is in interference fit with the second dynamic sealing ring, and the two are rotated and sealed.
According to the separation method of the piston-type biological liquid separation cup with inner core according to claim 9, the separation method comprises single treatment and continuous treatment;

The steps of the single treatment are:

a The centrifuge on the separation equipment drives the separation cup to rotate at high speed;

bUsing the peristaltic pump as the power, a certain amount of gradient liquid ficoll is transported to the hemispherical converging hole at the bottom of the inner core through the liquid inlet channel; under the action of centrifugal force, the gradient liquid climbs rapidly along the diversion channel to the annular stopper Plate, and then quickly climb into the separation chamber along the waist hole and drainage cone;

c Use a peristaltic pump to transport the biological fluid to be separated from the liquid inlet channel to the hemispherical gathering hole at the bottom of the inner core; the biological fluid will quickly enter the separation chamber along the diversion channel; when the biological fluid completely enters the separation cup, all the biological fluid The liquid will be located in the separation chamber; after a period of centrifugation, the heavier red blood cells will be at the outermost side of the separation chamber, and then the gradient liquid, buffy coat and plasma will be inward sequentially;

d Use the air pump to push the piston, so that the volume of the separation chamber gradually decreases, and the inner plasma and buffy coat are pushed out in turn; cooperate with the valve, color sensor, pipeline component sensor, component collection bag and waste liquid on the separation device bag to collect the required ingredients;

e When only red blood cells and gradient liquid are left in the separation chamber, the air pump pumps air in reverse, the actuating piston moves downward, and at the same time the centrifuge stops slowly; the liquid in the separation chamber will flow into the hemispherical concentrating hole under the action of gravity Under the action of the peristaltic pump, the remaining liquid is sucked away from the guide tube to complete the separation;

The steps of the continuous processing are:

The centrifuge on the A separation device drives the separation cup to rotate at high speed;

The B equipment is driven by a peristaltic pump to transport the biological fluid from the liquid inlet channel to the hemispherical gathering hole at the bottom of the inner core; under the action of centrifugal force, the biological fluid climbs rapidly along the diversion channel to the circular baffle, Then climb rapidly along the waist hole and the drainage cone to the separation cavity, and the cell fluid gradually fills the separation cavity; during the process of filling the separation cavity with the biological fluid, the cells in the biological fluid are distributed in the separation cavity under the action of centrifugal force. The outermost chamber, while the waste liquid is at the innermost side of the separation chamber;

C With the continuous input of biological fluid, the innermost waste liquid without cells in the separation chamber is squeezed out; when the biological liquid is completely input into the separation cup, the outer side of the separation chamber is cells, the inner side is waste liquid, and the sum of the volumes is the separation volume. The maximum dynamic volume of the cup;

DStart the air pump, push the piston, cooperate with the gravity sensor, optical sensor, valve and other components on the separation equipment, until there is a certain volume in the separation cup, stop pushing the piston;

E The centrifuge is stopped, and the air pump is started, and the air is pumped in reverse, so that the piston moves to the bottom; under the action of gravity, all the biological liquid in the separation cup is collected at the hemispherical flow hole at the bottom of the inner core; then the peristaltic pump is started , absorb the remaining biological fluid from the guide tube;

F suction a small amount of cleaning solution from the liquid outlet, and start the centrifuge to clean the liquid outlet, liquid outlet channel, separation chamber and diversion channel; then start the peristaltic pump again to suck the residual cells from the liquid inlet channel into the product bag.