CN115212931B - Platelet in-vitro release system and platelet production method - Google Patents
Platelet in-vitro release system and platelet production method Download PDFInfo
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- CN115212931B CN115212931B CN202110408609.2A CN202110408609A CN115212931B CN 115212931 B CN115212931 B CN 115212931B CN 202110408609 A CN202110408609 A CN 202110408609A CN 115212931 B CN115212931 B CN 115212931B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
The invention provides an in vitro platelet in vitro release system and a platelet preparation method, comprising the following steps: the device comprises a cover body, a cavity part, a raw material inlet, a product outlet and a driving liquid inlet, wherein the cavity part is arranged in the cover body, and the raw material inlet, the product outlet and the driving liquid inlet are all arranged on the surface of the cover body; the filter membrane comprises a filter membrane body and a plurality of filter holes arranged on the filter membrane body, wherein the filter holes are conical, the large openings of the filter holes face to the upper half cavity, the caliber of the large openings of the filter holes is 6-20 mu m, the caliber of the small openings of the filter holes is 2-6 mu m, and the thickness of the filter membrane body is 10-100 mu m; pressure adjusting device and flow adjusting device. The invention has the beneficial effects that: megakaryocyte enters the microfluidic chip, is trapped by the conical micropore array and deforms under the action of pressure, part of megakaryocyte is extruded outside the conical micropore, and the megakaryocyte releases platelets under the action of the shearing force of the parallel filter membrane and the turbulence generated by the microstructure. The batch production of platelets can be realized by uninterrupted injection of megakaryocytes.
Description
Technical Field
The invention belongs to the field of biological material manufacturing equipment, and particularly relates to an extracorporeal platelet release system and a platelet production method.
Background
Platelets have the important functions of stopping bleeding and clotting, promoting wound healing, participating in immune inflammatory reaction and the like, and various diseases such as bleeding and the like can be caused by thrombocytopenia or dysfunction. Platelets in clinical demand depend on volunteer blood donation, but the uncertainty of the number of blood donors and the like often cause insufficient platelet supply, so that the method has great clinical significance in the in-vitro mass production of functional platelets. Platelets are produced in vivo by megakaryocytes in bone marrow hematopoietic tissues, i.e., multifunctional hematopoietic stem cells in hematopoietic tissues undergo directional differentiation to form primitive megakaryocytes, which then develop into mature megakaryocytes, from which the pre-platelets extend and are released into blood vessels to form platelets. It has been demonstrated that cord blood hematopoietic stem cells, peripheral blood hematopoietic stem cells, and bone marrow hematopoietic stem cells can all be cultured in vitro and induced to produce megakaryocytes, ultimately producing platelets (Lee et al, 2014, strassl et al, 2016). The induction of megakaryocytes and platelets by pluripotent stem cells, particularly pluripotent induced stem cell bodies, has a number of unique advantages. For example, multipotent induced stem cells can give rise not only to megakaryocyte lines that can be expanded for a long period of time, but also to platelets that can prevent immune responses by genetic manipulation (Sugimoto et al 2017). In particular, megakaryocytes with a high amplifying ability and a high platelet yield can be obtained by introducing appropriate transcription factors and regulatory related mature transcription factors (Nakamura et al, 2014; moreau et al, 2016). Nevertheless, the specific mechanism of the platelet production process is currently unknown. Recent studies have also shown that megakaryocytes can circulate from the bone marrow to the lungs before releasing platelets. The designed platelet in vitro release device and method have defects, and various regulation parameters also need to be optimized.
Most of the existing devices are limited to megakaryocyte growth and platelet release in a static system, and the number of megakaryocytes and platelets obtained is very limited. Shear forces can be applied to mature megakaryocytes under flow conditions, thereby increasing the efficiency of platelet release. For example, blin et al designed bioreactors incorporate columnar arrays in microfluidic chip channels and modify their von Willebrand factor matrices to enhance interaction with mature megakaryocytes, which elongate and break under shear forces to increase platelet release (Blin et al 2016). Thon et al developed a microfluidic chip with two parallel channels using the principle of pressure difference. Megakaryocytes flowing in the first channel are brought into the space between the parallel channels by the fluid pressure differential, and trapped megakaryocytes extend their platelets into the second channel and release them (Thon et al, 2014). Pallotta et al use a hydrogel contractile, stretched silk fibroin to mimic a "spongy" bone marrow scaffold material, and then introduce mature megakaryocytes into the period and apply a force, thereby producing functional platelets (Pallotta et al, 2011). The above devices have a small fluid throughput such that the platelets produced are also limited.
Disclosure of Invention
In order to solve the technical problems, the invention provides an in-vitro platelet release system and a platelet production method.
The specific technical scheme is as follows:
An extracorporeal platelet release system, which differs in that it comprises:
The micro-fluidic chip comprises a cover body, a cavity body, a raw material inlet, a product outlet and a driving liquid inlet, wherein the cavity body is arranged in the cover body, and the raw material inlet, the product outlet and the driving liquid inlet are all arranged on the surface of the cover body;
The filter membrane is arranged on the cavity part, the cavity part is divided into an upper half cavity part and a lower half cavity part by the filter membrane, the raw material inlet is communicated with the upper half cavity part, the product outlet is communicated with the lower half cavity part, the filter membrane comprises a filter membrane body and a plurality of filter holes arranged on the filter membrane body, the filter holes are conical, and the big openings of the filter holes face the upper half cavity part;
The pressure adjusting device can respectively adjust the pressure of the upper half cavity part and the pressure of the lower half cavity part;
And
And the flow regulating device can respectively regulate the flow of the liquid entering the upper half cavity part and the lower half cavity part.
Compared with the prior art, the invention has the beneficial effects that: passing megakaryocyte through a conical filter membrane with a conical through hole, extruding through the conical through hole, deforming under the action of fluid pressure, and splitting under the action of shearing force outside the conical micropore small aperture to generate high-yield platelets; meanwhile, the design of the isolation cavity is easier to collect products.
Further, the large aperture of the filtering hole is 6-20 mu m, the small aperture is 2-6 mu m, and the thickness of the filtering film body is 10-100 mu m;
further, the distribution density number of the filter holes on the filter membrane body is not less than 30 ten thousand/cm 2.
Further, the turbulence piece is arranged in the lower cavity half, and comprises a turbulence piece body and a plurality of barriers, wherein the barriers are distributed on the turbulence piece body.
The beneficial effects of adopting the further technical scheme are that: when the driving liquid entering from the driving liquid inlet passes through the obstacle, turbulent flow is formed, and the uninterrupted progress of the fluid shearing force is further ensured.
Further, the turbulence member is disposed on the bottom surface of the lower cavity half and aligned with the filter membrane.
Further, the pressure regulating device is installed in a communication system with the upper half cavity portion and the lower half cavity portion, and the flow regulating device is installed in a communication system with the upper half cavity portion and the lower half cavity portion.
Further, the surface of the cover body is also provided with a pressure control port, the driving liquid inlet and the product outlet are communicated with the lower half cavity part through a first channel, and the raw material inlet and the pressure control port are communicated with the upper half cavity part through a second channel.
The beneficial effects of adopting the further technical scheme are that: the system pressure stability can be maintained by controlling the closing or opening of the pressure control port.
Further, the widths of the first channel and the second channel are 1-5 mm, and the heights of the first channel and the second channel are 0.2-1 mm.
Further, the barrier is a plane orientation protrusion, the plane orientation protrusion comprises a long arm and a short arm, the long arm and the short arm form an included angle, and the included angle direction of the long arm and the short arm points to the driving liquid inlet;
Further, the thickness of the long arm and the short arm is 0.2 mm-1 mm, the width of the long arm and the short arm is 0.1 mm-1 mm, the length of the long arm is 0.5 mm-2 mm, the length of the short arm is 0.2 mm-1 mm, and the included angle between the long arm and the short arm is 45 degrees-90 degrees.
The beneficial effects of adopting the further technical scheme are that: the obstacle adopts the design of the structural parameters, so that the turbulence piece can form turbulence in the lower half cavity part.
Further, the included angle between the long arm and the short arm is 60-90 degrees.
The beneficial effects of adopting the further technical scheme are that: the turbulent mixing effect can be better by adopting the included angle range.
Further, the lid is including mutually supporting upper cover body and lower cover body, the upper cover body with the inside of lower cover body respectively contains a part of cavity portion, the upper cover body with be equipped with the mantle layer between the lower cover body, the mantle layer sets up in the cavity both sides and includes mantle layer and lower mantle layer, go up the mantle layer with upper cover body coupling, lower mantle layer with lower cover body coupling.
The beneficial effects of adopting the further technical scheme are that: the split structure design of the upper cover body and the lower cover body can facilitate the manufacture and the replacement of the filter membrane core component, and the design of the soft membrane layer can ensure the tightness.
Further, the upper soft film layer and the upper cover body are in vacuum bonding, and the lower soft film layer and the lower cover body are in vacuum bonding.
Further, the platelet release system further comprises a raw material container, a platelet collection container, a driving liquid container and a pressure control container, wherein the raw material container is connected with the raw material inlet through a first pipeline, the platelet collection container is connected with the product outlet through a second pipeline, the driving liquid container is connected with the driving liquid inlet through a third pipeline, and the pressure control container is connected with the pressure control opening through a fourth pipeline.
Further, the pressure regulating device comprises a pressure regulator arranged on the first pipeline, the second pipeline and the fourth pipeline, and the flow regulating device comprises a flow regulator arranged on the first pipeline and the second pipeline.
Further, the preparation raw materials of the filter membrane comprise polymers and photosensitive curing glue and/or thermosetting glue.
Further, the filter membrane further comprises a reinforcing ring arranged along the edge of the filter membrane body.
Further, the diameters of the upper half cavity part and the lower half cavity part are 10 mm-20 mm, and the heights of the upper half cavity part and the lower half cavity part are 0.2 mm-1 mm.
Further, the upper cover body and the lower cover body are made of polycarbonate subjected to oxygen plasma surface treatment as a main material, the upper microfluidic chip is bonded with the upper soft film layer, the lower cover body is bonded with the lower soft film layer, and the upper cover body and the lower cover body are sealed through the bonding of the upper soft film layer and the lower soft film layer.
Further, the upper soft film layer and the lower soft film layer are made of PDMS.
The method for producing the platelets by using the platelet in-vitro release system is characterized in that megakaryocytes enter the upper half cavity part from the raw material inlet, are extruded through the filtering holes, pass through the filtering holes, simultaneously pass through the driving liquid inlet, pass through the driving liquid to carry out fluid shearing, and finally collect the products entering the lower half cavity part.
Further, the pressure of megakaryocyte entering the upper half cavity is 5 mbar-20 mbar, the pressure of fluid in the lower half cavity passing through the filter holes is 10 mbar-100 mbar, and the pressure of 100 mbar-400 mbar drives the culture medium to enter the lower half cavity, and the flow rate is 5 ml/min-15 ml/min.
Drawings
FIG. 1 is a schematic diagram of an embodiment of the platelet in vitro release system of example 1;
FIG. 2 is a top view of a filter membrane;
FIG. 3 is a cross-sectional view of FIG. 2;
FIG. 4 is a schematic diagram of an embodiment of a microfluidic chip according to example 1;
FIG. 5 is an enlarged view of a portion of the turbulence member at a bottom angle;
FIG. 6 is a schematic diagram of an embodiment of a microfluidic chip according to example 1;
fig. 7 is a schematic view of the microfluidic chip upper cover body shown in fig. 6;
fig. 8 is a schematic view of a lower cover body of the microfluidic chip shown in fig. 6;
FIG. 9 is a schematic diagram of an embodiment of the platelet in vitro release system of example 1;
FIG. 10 is a schematic diagram of the principle of megakaryocyte platelet production;
FIG. 11 shows the residual condition of megakaryocytes after passing through the filter pores;
FIG. 12 is a fluorescent labeling of the cytoskeleton after deformation of the submembranous megakaryocytes;
FIG. 13 is a view of a fluorescent label of the recovered platelets;
In the drawings, the list of components represented by the various numbers is as follows:
The micro-fluidic chip-1, a cover body-101, a cavity body-102, an upper cavity body-102 a, a lower cavity body-102 b, a raw material inlet-1011, a product outlet-1012, a driving liquid inlet-1013, a pressure control port-1014, an upper cover body-1015, a lower cover body-1016, a first channel-103, a second channel-104, a filter membrane-2, a filter membrane body-201, a filter hole-202, a reinforcing ring-203, a turbulence member-3, a turbulence member body-301, the device comprises a lower side face-301 a, an obstacle-302, a long arm-3021, a short arm-3022, a soft membrane layer-4, an upper soft membrane layer-401, a lower soft membrane layer-402, a raw material container-5, a platelet collecting container-6, a driving liquid container-7, a pressure control container-8, a first pipeline-9, a second pipeline-10, a third pipeline-11, a fourth pipeline-12, a pressure regulator-13, a flow regulator-14, a luer orifice-15, megakaryocytes-16 and platelets-17.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
Example 1
The present embodiment provides an extracorporeal platelet release system, specifically as shown in fig. 1, comprising:
The microfluidic chip 1, the microfluidic chip 1 comprises a cover body 101, a cavity 102, a raw material inlet 1011, a product outlet 1012 and a driving liquid inlet 1013, wherein the cavity 102 is arranged in the cover body 101, and the raw material inlet 1011, the product outlet 1012 and the driving liquid inlet 1013 are all arranged on the surface of the cover body 101;
a filter membrane 2 arranged in the cavity 102, wherein the filter membrane 2 divides the cavity 102 into an upper half cavity 102a and a lower half cavity 102b, a raw material inlet 1011 is communicated with the upper half cavity 102a, a product outlet 1012 is communicated with the lower half cavity 102b, and the filter membrane comprises a filter membrane body 201 and a plurality of filter holes 202 arranged on the filter membrane body 201;
The pressure adjusting device can be used for adjusting and monitoring the pressure of the upper half cavity part and the lower half cavity part, and the equipment and the installation mode for achieving the functions of the pressure adjusting device are not limited, and the existing pressure adjusting device can be selected to be respectively installed on the upper half cavity part and the lower half cavity part or be respectively installed in a communication system of the upper half cavity part and the lower half cavity part as shown in fig. 1.
The flow rate adjusting device can be used for adjusting and monitoring the flow rate of the upper half cavity part and the lower half cavity part, and the equipment and the installation mode for achieving the functions of the flow rate adjusting device are not limited, and the existing pressure adjusting device can be selected to be respectively installed in the upper half cavity part and the lower half cavity part, or can be selected to be respectively installed in a communication system of the upper half cavity part and the lower half cavity part as shown in fig. 1.
In this embodiment, as shown in fig. 2 to 3, the filter membrane structure is shown in fig. 2 to 3, the filter hole 202 is tapered, the large opening of the filter hole 202 faces to the upper half cavity, the large opening caliber of the filter hole 202 is 6 to 20 μm, the small opening caliber is 2 to 6 μm, the thickness of the filter membrane body is 10 μm to 100 μm, the large opening can ensure the capturing rate of megakaryocyte, and the small opening micropores can control the deformation of megakaryocyte on one hand, and continuously generate platelets through continuous extrusion on the other hand, so that the yield of platelets is further improved. Compared with the traditional straight-tube filter membrane, the conical-hole filter membrane can avoid the blockage of the filter holes. The number of distribution density of the filter holes 202 on the filter membrane body 201 is not less than 30 ten thousand/cm 2, the more the number of the filter holes is distributed, the higher the platelet yield is, the material of the filter membrane 2 is selected to be a material which is not easy to deform, such as PEGDA, PLGA and the like, the material selected in the embodiment is PEGDA, in order to facilitate the operability of the filter membrane 2, in the embodiment, a reinforcing ring 203 is arranged along the periphery of the filter membrane body 201, the size parameter of the reinforcing ring can be flexibly adjusted according to the size, thickness and material of the filter membrane, and in the embodiment, the inner diameter of the reinforcing ring 203 is 5 mm-18 mm, and the outer diameter is 12-22 mm.
In this embodiment, the diameter of the entire cavity 102 is 20mm to 40mm, and the heights of the upper cavity half 102a and the lower cavity half 102b are 0.2mm to 1mm.
In order to further increase the yield of platelets, the inner bottom surface of the lower cavity half 102b is provided with a turbulence member 3, the turbulence member 3 comprises a turbulence member body 301 and a plurality of barriers 302, and the barriers 302 are distributed on the turbulence member body 301. The barrier 302 is mainly aimed at providing resistance to the megakaryocyte after being extruded through the filter hole, forming turbulence during the flowing process, ensuring continuous provision of fluid shear force, further improving the yield, and the specific structure of the barrier is not limited based on the above purpose. In this embodiment, as shown in fig. 4, turbulence members 3 are disposed on two sides of the lower portion of the filter membrane 2. The barrier 302 is a planar orientation protrusion, and the specific structure of the planar orientation protrusion is shown in fig. 5, where the planar orientation protrusion includes a long arm 3021 and a short arm 3022, and the long arm 3021 and the short arm 3022 form an included angle, and the direction of the included angle points to the driving liquid inlet 1013. In this embodiment, the thickness of the long arm 3021 and the short arm 3022 is 0.2 to 1mm, the width of the long arm 3021 and the short arm 3022 is 0.1 to 1mm, the length of the long arm 3021 is 0.5 to 2mm, the length of the short arm 3022 is 0.2 to 1mm, the included angle between the long arm 3021 and the short arm 3022 is 45 ° to 90 °, and turbulence is ensured in the liquid in the lower half cavity.
Further, the included angle between the long arm 3021 and the short arm 3022 is between 60 ° and 90 °, so as to ensure that the liquid in the lower half cavity generates turbulence, and the included angle in this range can make the turbulence mixing efficiency higher, and in this embodiment, the included angle between the long arm and the short arm is between 70 °.
In this embodiment, as shown in fig. 4, in order to further improve the pressure stability of the upper half cavity 102a, a pressure control port 1014 is further provided on the surface of the cover 101, the raw material inlet 1011 and the pressure control port 1014 are communicated with the upper half cavity 102a and not with the lower half cavity 102b through the second channel 104, and the pressure control port 1014 can be freely closed or opened, so as to maintain the stable pressure of the whole system. In the present embodiment, the raw material inlet 1011 and the product outlet 1012 are disposed on one side of the cavity, the driving liquid inlet 1013 and the pressure control port 1014 are disposed on the other side of the cavity, and the product outlet 1012 and the driving liquid inlet 1013 are communicated with the lower cavity 102b and not communicated with the upper cavity 102a through the first channel 103.
In order to increase the convenience of manufacturing the microfluidic chip and the subsequent reusability, the microfluidic chip is manufactured by adopting a split structure, specifically, as shown in fig. 6 to 8, the cover body 101 includes an upper cover body 1015 and a lower cover body 1016 that are mutually matched, the interiors of the upper cover body 1015 and the lower cover body 1016 respectively include a part of the cavity 102, in this embodiment, the upper cavity 102a is disposed in the upper cover body 1015, the lower cavity 102b is disposed in the lower cover body 1016, and the filter membrane 2 is also disposed in the interior of the lower cover body 1016. In order to increase the tightness of the upper cover body 1015 and the lower cover body 1016, a soft film layer 4 is arranged between the upper cover body 1015 and the lower cover body 1016, the soft film layer 4 is arranged at two sides of the cavity 102 and comprises an upper soft film layer 401 and a lower soft film layer 402, the upper soft film layer 401 is connected with the upper cover body 1015, and the lower soft film layer 402 is connected with the lower cover body 1016. In this embodiment, the upper soft membrane layer 401 and the lower soft membrane layer 402 are made of PDMS, the upper cover body 1015 and the lower cover body 1016 are made of polycarbonate subjected to oxygen plasma surface treatment to improve the subsequent bonding property with the PDMS soft membrane, the upper cover body 1015 is vacuum bonded with the upper soft membrane layer 401, the lower cover body 1016 is vacuum bonded with the lower soft membrane layer 402, and the upper cover body 1015 is vacuum bonded and sealed connected with the lower cover body 1016 through the upper soft membrane layer 401 and the lower soft membrane layer 402.
In this embodiment, for convenience of continuous use of the platelet delivery system, as shown in fig. 9, the platelet delivery system further comprises a raw material container 5, a platelet collection container 6, a driving liquid container 7, and a pressure control container 8, wherein the raw material container 5 is connected with the raw material inlet 1011 through a first pipe 9, the platelet collection container 6 is connected with the product outlet 1012 through a second pipe 10, the driving liquid container 7 is connected with the driving liquid inlet 1013 through a third pipe 11, and the pressure control container 8 is connected with the pressure control port 1014 through a fourth pipe 12. Specifically, luer fittings (not shown) are used as connectors to connect the individual containers, with luer fittings 15 being installed at the respective ports. A pressure regulator 13 and a flow regulator 14 connected to the source container 5, a pressure regulator 13 and a flow regulator 14 connected to the platelet collection container 6, and a flow regulator 14 connected to the fourth conduit 12. In the present embodiment, the pressure regulator 13 is an electric pressure regulating valve, and the flow regulator 14 is a gas-liquid electromagnetic valve.
Example 2
This embodiment provides a method for producing platelets by using the platelet delivery system shown in fig. 9 of embodiment 1, the schematic diagram of which is shown in fig. 10, and specifically includes:
The pluripotent stem cell-induced megakaryocyte (purchased from Thermofisher company) in IMDM/F12 (3:1 v/v) culture medium is added into the upper half cavity from the raw material inlet 1011, the upper half cavity is applied with pressure of 5 mbar-20 mbar to capture the cell in the filter hole, the upper cavity liquid can be removed at the same time when the pressure is applied to capture the cell in the filter hole, the cell membrane is broken due to the too strong pressure, and the cell body is not effectively immobilized in the filter hole due to the too weak pressure. And increasing the pressure of the upper half cavity to 10 mbar-100 mbar, extruding cells, wherein the cell membrane is crushed by the excessively strong pressure, so that the cells are cracked, and the cell body cannot be effectively extruded by the excessively weak pressure, so that the shearing force is unfavorable for acting on the cell body to generate platelets. Then, by applying a pressure of 100-400mbar to the lower cavity half 102b, the culture medium of IMDM/F12 (3:1v/v) is driven to flow in, the pressure difference drives the flow speed to further control the shear force, the pressure is too weak, the shear force is too small, the platelet production efficiency is affected, the too strong pressure exceeds the upper cavity maintenance pressure, the liquid possibly gushes out to the upper cavity, the cell body is separated from the filter hole, and the experiment fails. The flow rate is 5-15 ml/min, the exposed megakaryocyte 16 is sheared, the flow rate determines the shearing force, the flow rate is too high, the shearing force activates the blood platelet, and the activity of the blood platelet is changed; too weak a flow rate, the platelet production efficiency is reduced, and finally the platelets 17 are collected.
Microscopic observation is performed on the above process, wherein fig. 11 shows the residual condition of megakaryocytes after passing through the filter hole, and shows that megakaryocytes are successfully captured at the filter hole; FIG. 12 is a fluorescent label of the cytoskeleton of a submembrane megakaryocyte after being deformed by force, 12a is the shape of the megakaryocyte before being deformed, and 12b is the shape of the megakaryocyte after being deformed by extrusion and acted by a shearing force, wherein the deformation of the megakaryocyte in the figure shows that the megakaryocyte is extruded at a filter hole; FIG. 13 is a fluorescence labeling plot (CD41+) of the recovered platelets, showing a size of 2-4 microns, consistent with platelet size, through megakaryocytes of the filter wells to produce platelets.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (7)
1. An extracorporeal platelet release system comprising:
The micro-fluidic chip comprises a cover body, a cavity body, a raw material inlet, a product outlet and a driving liquid inlet, wherein the cavity body is arranged in the cover body, and the raw material inlet, the product outlet and the driving liquid inlet are all arranged on the surface of the cover body;
The filter membrane is arranged on the cavity part, the cavity part is divided into an upper half cavity part and a lower half cavity part by the filter membrane, the raw material inlet is communicated with the upper half cavity part, the product outlet and the driving liquid inlet are communicated with the lower half cavity part, the filter membrane comprises a filter membrane body and a plurality of filter holes arranged on the filter membrane body, the filter holes are conical, and the large openings of the filter holes face the upper half cavity part;
The pressure adjusting device can respectively adjust the pressure of the upper half cavity part and the pressure of the lower half cavity part;
The flow regulating device can respectively regulate the flow of liquid entering the upper half cavity part and the lower half cavity part;
the inner part of the lower half cavity is provided with a turbulence piece, and the turbulence piece comprises a turbulence piece body and a plurality of barriers distributed on the turbulence piece body;
The surface of the cover body is also provided with a pressure control port, the driving liquid inlet and the product outlet are communicated with the lower half cavity part through a first channel, and the raw material inlet and the pressure control port are communicated with the upper half cavity part through a second channel;
the cover body comprises an upper cover body and a lower cover body which are mutually matched, wherein the interiors of the upper cover body and the lower cover body respectively comprise a part of the cavity body, a soft film layer is arranged between the upper cover body and the lower cover body, and the soft film layers are arranged on two sides of the cavity body; the soft film layer comprises an upper soft film layer and a lower soft film layer, wherein the upper soft film layer is connected with the upper cover body, and the lower soft film layer is connected with the lower cover body.
2. The extracorporeal platelet release system of claim 1, wherein the barrier is a planar directional protrusion comprising a long arm and a short arm, the long arm and the short arm forming an included angle of 45 ° to 90 °, the included angle being directed toward the driving liquid inlet.
3. The extracorporeal platelet release system of claim 1, wherein the upper soft membrane layer is vacuum bonded to the upper cap body and the lower soft membrane layer is vacuum bonded to the lower cap body.
4. The extracorporeal platelet release system of claim 1, further comprising a raw material container, a platelet collection container, a drive liquid container, and a pressure control container, wherein the raw material container is connected to the raw material inlet through a first conduit, and the platelet collection container is connected to the product outlet through a second conduit; the driving liquid container is connected with the driving liquid inlet through a third pipeline, and the pressure control container is connected with the pressure control port through a fourth pipeline.
5. The extracorporeal platelet release system of claim 1, wherein the filter membrane further comprises a stiffening ring disposed along an edge of the filter membrane body.
6. A method of producing platelets using the platelet in vitro release system according to any one of claims 1 to 5, wherein megakaryocytes are introduced into said upper half-cavity from said raw material inlet, pressed through said filter holes, and simultaneously subjected to fluid shear from said driving liquid inlet while passing through said filter holes, and finally the product introduced into said lower half-cavity is collected.
7. The method according to claim 6, wherein megakaryocytes enter the upper half-chamber at a pressure of 5 to 20mbar, the fluid in the lower half-chamber passing through the filter holes at a pressure of 10 to 100mbar, and the medium is driven into the lower half-chamber at a pressure of 100 to 500mbar at a flow rate of 5 to 15mL/min.
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