CN115212931A - Platelet in-vitro release system and platelet production method - Google Patents

Platelet in-vitro release system and platelet production method Download PDF

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Publication number
CN115212931A
CN115212931A CN202110408609.2A CN202110408609A CN115212931A CN 115212931 A CN115212931 A CN 115212931A CN 202110408609 A CN202110408609 A CN 202110408609A CN 115212931 A CN115212931 A CN 115212931A
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cover body
cavity part
platelet
driving liquid
megakaryocytes
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CN115212931B (en
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黄博鑫
石剑
汪莉
陈勇
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WUHAN JIEGUAN BIO-TECHNOLOGY CO LTD
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WUHAN JIEGUAN BIO-TECHNOLOGY CO LTD
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • B01L3/502753Containers 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
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • B01L3/502761Containers 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
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0647Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652Sorting or classification of particles or molecules
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/06Auxiliary integrated devices, integrated components
    • B01L2300/0681Filter

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  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
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Abstract

The invention provides an in vitro platelet release system and a platelet preparation method, which comprise the following steps: the cover body, the cavity part, the raw material inlet, the product outlet and the driving liquid inlet are 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, the filter holes are conical, the large openings of the filter holes face the upper half cavity part, the caliber of the large openings of the filter holes is 6-20 mu m, the caliber of the small openings is 2-6 mu m, and the thickness of the filter membrane body is 10-100 mu m; pressure regulating device and flow regulator. The invention has the beneficial effects that: the megakaryocytes enter the microfluidic chip, are trapped by the conical micropore array and deform under the action of pressure, part of the megakaryocytes are extruded outside the conical micropores, and the megakaryocytes release platelets under the action of turbulence generated by the shearing force of the parallel filter membrane and the microstructure. The megakaryocytes can be uninterruptedly injected to realize the mass production of the platelets.

Description

Platelet in-vitro release system and platelet production method
Technical Field
The invention belongs to the field of biological material manufacturing equipment, and particularly relates to a platelet in-vitro release system and a platelet production method.
Background
Platelets have important functions of stopping bleeding, clotting blood, promoting wound healing, participating in immune inflammatory reaction and the like, and thrombocytopenia or dysfunction can cause various diseases such as bleeding. Platelets in clinical demand depend on blood donation of volunteers, but platelet storage period is short, uncertainty of the number of blood donors and the like often cause insufficient platelet supply, so that the mass production of functional platelets in vitro has great clinical significance. The platelets are produced in vivo by the megakaryocytes in the bone marrow hematopoietic tissue, i.e., the multifunctional hematopoietic stem cells in the hematopoietic tissue undergo directional differentiation to form primitive megakaryocytes, which then progress to mature megakaryocytes, and the anterior platelets extend from the megakaryocytes into the blood vessels and are released to produce platelets. It has been demonstrated that cord blood, peripheral blood and bone marrow hematopoietic stem cells can all be cultured in vitro and induced to produce megakaryocytes and, ultimately, platelets (Lee et al, 2014, strassel et al, 2016). The induction of megakaryocytes and platelets with pluripotent stem cells, and in particular with pluripotent-induced stem cell bodies, has many unique advantages. For example, pluripotent-inducing stem cells can be used to obtain not only a megakaryocyte cell line that can be expanded for a long period of time, but also platelets that can prevent immune responses by genetic manipulation (Sugimoto et al 2017). In particular, megakaryocytes with high amplification capacity and high platelet yield can be obtained by introducing appropriate transcription factors and regulating related mature transcription factors (Nakamura et al, 2014; moreau et al, 2016). Nevertheless, the specific mechanism of the platelet production process is not currently known. Recent studies have also shown that megakaryocytes release platelets after circulation from the bone marrow to the lungs. The designed in-vitro platelet release device and method have defects, and various control 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 may be applied to the mature megakaryocytes under flow conditions, thereby increasing the efficiency of platelet release. For example, the bioreactor designed by Blin et al integrates a columnar array in a microfluidic chip channel and modifies its von Willebrand factor matrix to enhance interaction with mature megakaryocytes, which elongate and fragment under shear forces to increase platelet release (Blin et al, 2016). Thon et al developed a microfluidic chip with two parallel channels using the pressure differential principle. Under the effect of the fluid pressure differential, megakaryocytes flowing in the first channel are carried into the spaces between the parallel channels, and trapped megakaryocytes extend their pre-platelets to the second channel and release the platelets (Thon et al, 2014). Pallotta et al used stretched silk fibroin with hydrogel contractility to mimic a "sponge-like" bone marrow scaffold material, and then introduced mature megakaryocytes into the process and applied a force that produced functional platelets (Pallotta et al, 2011). The above-described apparatus has a small fluid throughput such that platelets produced are also limited.
Disclosure of Invention
In order to solve the technical problems, the invention provides a platelet in-vitro release system and a platelet production method.
The specific technical scheme is as follows:
an in vitro platelet delivery system, comprising:
the micro-fluidic chip 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 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 large openings of the filter holes face the upper half cavity part;
a pressure regulating device that can respectively regulate the pressure of the upper half chamber portion and the lower half chamber portion;
and
and the flow regulating device can respectively regulate the liquid flow entering the upper half cavity part and the liquid flow entering the lower half cavity part.
Compared with the prior art, the invention has the beneficial effects that: the megakaryocytes pass through a conical filter membrane with a conical through hole, are extruded by the conical through hole, deform under the action of fluid pressure, and are extruded to the outside of the small aperture of the conical micropore and split under the action of shearing force to generate high-yield platelets; meanwhile, due to the design of the isolation cavity, products can be collected more easily.
Furthermore, the aperture of the large opening of the filtering hole is 6-20 μm, the aperture of the small opening is 2-6 μm, and the thickness of the filter membrane body is 10-100 μm;
furthermore, the distribution density of the filter holes on the filter membrane body is not less than 30 ten thousand/cm 2
Further, the interior of the lower half cavity part is provided with a turbulence piece, the turbulence piece comprises a turbulence piece body and a plurality of obstacles, and the obstacles are distributed on the turbulence piece body.
The beneficial effect of adopting the further technical scheme is that: when the driving liquid entering from the driving liquid inlet passes through the barrier, turbulent flow is formed, and uninterrupted operation of the fluid shearing force is further ensured.
Further, the turbulence piece is arranged on the bottom surface inside the lower half cavity and is aligned with the filter membrane.
Further, the pressure adjusting device is installed in a communication system with the upper half chamber portion and with the lower half chamber portion, and the flow adjusting device is installed in a communication system with the upper half chamber portion and with the lower half chamber portion.
Furthermore, 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 effect of adopting the further technical scheme is that: the pressure stability of the system can be maintained by controlling the closing or opening of the pressure control port.
Further, the width of the first channel and the second channel is 1 mm-5 mm, and the height of the first channel and the second channel is 0.2 mm-1 mm.
Furthermore, the barrier is a plane directional projection, the plane directional projection comprises a long arm and a short arm, the long arm and the short arm form an included angle, and the direction of the included angle 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 45-90 degrees.
The beneficial effect of adopting the further technical scheme is that: the obstacle adopts the design of the structural parameters to enable the turbulence piece to form turbulence in the lower cavity part.
Furthermore, the included angle between the long arm and the short arm is 60-90 degrees.
The beneficial effect of adopting the further technical scheme is that: adopt above-mentioned contained angle scope can make turbulent flow mixing effect better.
Further, the lid includes upper cover body and lower cover body of mutually supporting, the upper cover body reaches the inside of lower cover body contains respectively partly of cavity portion, the upper cover body reaches be equipped with soft rete between the lower cover body, soft rete sets up in the cavity both sides and includes soft rete and lower soft rete, go up soft rete with this body coupling of upper cover, lower soft rete with this body coupling of lower cover.
The beneficial effect of adopting the further technical scheme is that: the split structure design of the upper cover body and the lower cover body can facilitate the manufacture and the replacement of the core part of the filter membrane, and the design of the soft membrane layer can ensure the sealing property.
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, platelet release system still includes raw materials container, platelet collection container, drive liquid container and accuse pressure container, raw materials container with the raw materials import is through first pipe connection, platelet collection container pass through the second pipeline with product exit linkage, drive liquid container with drive liquid import passes through the third pipe connection, accuse pressure container with accuse pressure mouthful passes through the fourth pipe connection.
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 a polymer and photosensitive curing glue and/or thermosetting glue.
Further, the filter membrane still includes along the reinforcement ring that sets up along the border of 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 prepared by taking 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 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 filter holes, pass through the filter holes, are introduced from the driving liquid inlet to carry out fluid shearing, and finally, a product entering the lower half cavity part is collected.
Furthermore, the pressure of the megakaryocytes entering the upper half cavity is 5mbar to 20mbar, the pressure of the fluid in the lower half cavity passing through the filter hole is 10mbar to 100mbar, the culture medium is driven to enter the lower half cavity by the pressure of 100mbar to 400mbar, and the flow rate is 5ml/min to 15ml/min.
Drawings
FIG. 1 is a schematic diagram of one embodiment of the in vitro platelet release system of example 1;
FIG. 2 is a top view of the filter membrane;
FIG. 3 is a cross-sectional view of FIG. 2;
FIG. 4 is a schematic diagram of one embodiment of the microfluidic chip of example 1;
FIG. 5 is a partial enlarged view of the turbulence member from a bottom view;
FIG. 6 is a schematic diagram of one embodiment of the microfluidic chip of example 1;
FIG. 7 is a schematic view of the upper cover body of the microfluidic chip shown in FIG. 6;
FIG. 8 is a schematic view of the lower cap body of the microfluidic chip shown in FIG. 6;
FIG. 9 is a schematic diagram of one embodiment of the in vitro platelet release system of example 1;
FIG. 10 is a schematic diagram of the principle of platelet production by megakaryocytes;
FIG. 11 is the residue of megakaryocytes after passing through the filtration pores;
FIG. 12 is a fluorescent labeling diagram of cytoskeleton after the deformation of submembrane megakaryocytes under stress;
FIG. 13 is a fluorescent labeling chart of recovered platelets;
in the drawings, the components represented by the respective reference numerals are listed below:
the device comprises a microfluidic chip-1, a cover body-101, a cavity part-102, an upper half cavity part-102 a, a lower half cavity part-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, a lower side surface-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 mother luer-15, a giant cell-16 and a platelet-17.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
Example 1
The present embodiment provides an in vitro platelet release system, as specifically shown in fig. 1, including:
the micro-fluidic chip 1 comprises a cover body 101, a cavity part 102, a raw material inlet 1011, a product outlet 1012 and a driving liquid inlet 1013, wherein the cavity part 102 is arranged inside 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;
the filter membrane 2 is arranged in the cavity part 102, the cavity part 102 is divided into an upper half cavity part 102a and a lower half cavity part 102b by the filter membrane 2, the raw material inlet 1011 is communicated with the upper half cavity part 102a, and the product outlet 1012 is communicated with the lower half cavity part 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 intensity of the upper half cavity part and the lower half cavity part, the equipment for achieving the functions and the installation mode are not limited, the existing pressure adjusting device can be selected to be respectively installed on the upper half cavity part and the lower half cavity part, and the existing pressure adjusting device can also 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 regulating device can be used for regulating and monitoring the flow of the upper half cavity part and the lower half cavity part, the equipment for achieving the functions and the installation mode are not limited, the existing pressure regulating devices can be selected to be respectively installed on the upper half cavity part and the lower half cavity part, and the existing pressure regulating devices can also 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 figure 1.
In this embodiment, the filter membrane structureAs shown in fig. 2 to 3, the filtering pores 202 are tapered, the large openings of the filtering pores 202 face the upper half cavity portion, the apertures of the large openings of the filtering pores 202 are 6 to 20 μm, the apertures of the small openings are 2 to 6 μm, and the thickness of the filter membrane body is 10 μm to 100 μm. Compared with the traditional straight-tube filter membrane, the conical-hole filter membrane can avoid the blockage of the filter holes. The distribution density of the filter holes 202 on the filter membrane body 201 is not less than 30 ten thousand/cm 2 In the embodiment, the material of the filter membrane 2 is selected from non-deformable materials, such as PEGDA, PLGA, etc., 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 disposed 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, in the embodiment, the inner diameter of the reinforcing ring 203 is 5mm to 18mm, and the outer diameter is 12mm to 22mm.
In the present embodiment, the diameter of the entire cavity 102 is 20mm to 40mm, and the heights of the upper half cavity 102a and the lower half cavity 102b are both 0.2mm to 1mm.
In order to further improve the yield of the platelets, the turbulence member 3 is arranged on the bottom surface of the lower cavity half part 102b, the turbulence member 3 comprises a turbulence member body 301 and a plurality of obstacles 302, and the obstacles 302 are distributed on the turbulence member body 301. The obstacle 302 is mainly used to provide resistance to flow of megakaryocytes after being squeezed through the filtering holes, so as to form turbulent flow, thereby ensuring continuous provision of fluid shear force, and further improving yield. In the present embodiment, as shown in fig. 4, two turbulence members 3 are respectively disposed on two sides below the filter membrane 2. The obstruction 302 is a planar orientation projection, and as shown in fig. 5, the planar orientation projection includes a long arm 3021 and a short arm 3022, and the long arm 3021 and the short arm 3022 form an angle, and the angle is directed toward 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, and the included angle between the long arm 3021 and the short arm 3022 is 45 ° to 90 °, so that the turbulent flow of the liquid in the lower half chamber is ensured.
Further, the included angle between the long arm 3021 and the short arm 3022 is 60 ° to 90 °, so as to ensure that the liquid in the lower cavity generates turbulent flow, and the included angle in this range can improve the turbulent mixing efficiency, and in this embodiment, the included angle between the long arm and the short arm is 70 °.
In this embodiment, as shown in fig. 4, in order to further improve the pressure stability of the upper-half chamber 102a, a pressure control opening 1014 is further formed on the surface of the cover 101, the raw material inlet 1011 and the pressure control opening 1014 are communicated with the upper-half chamber 102a through the second channel 104 and are not communicated with the lower-half chamber 102b, and the pressure control opening 1014 can be freely closed or opened, so as to maintain the stable pressure of the whole system. In this embodiment, the raw material inlet 1011 and the product outlet 1012 are disposed on one side of the chamber portion, the driving liquid inlet 1013 and the pressure control port 1014 are disposed on the other side of the chamber portion, and the product outlet 1012 and the driving liquid inlet 1013 are communicated with the lower half chamber portion 102b and not communicated with the upper half chamber portion 102a through the first passage 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 which are mutually matched, the insides of the upper cover body 1015 and the lower cover body 1016 respectively include a part of the cavity portion 102, in this embodiment, the upper half cavity portion 102a is disposed inside the upper cover body 1015, the lower half cavity portion 102b is disposed inside the lower cover body 1016, and the filter membrane 2 is also disposed inside the lower cover body 1016. In order to increase the sealing performance between the upper cap body 1015 and the lower cap body 1016, the soft film layer 4 is disposed on two sides of the cavity portion 102 and includes an upper soft film layer 401 and a lower soft film layer 402, the upper soft film layer 401 is connected to the upper cap body 1015, and the lower soft film layer 402 is connected to the lower cap body 1016. In this embodiment, the upper soft film layer 401 and the lower soft film layer 402 are made of PDMS, the upper cap body 1015 and the lower cap body 1016 are made of polycarbonate subjected to oxygen plasma surface treatment as a main material to improve the subsequent bonding property with the PDMS soft film, the upper cap body 1015 and the upper soft film layer 401 are vacuum bonded, the lower cap body 1016 and the lower soft film layer 402 are vacuum bonded, and the upper cap body 1015 and the lower cap body 1016 are hermetically connected through the vacuum bonding of the upper soft film layer 401 and the lower soft film layer 402.
In order to facilitate the continuous use of the platelet releasing system in this embodiment, as shown in fig. 9, the platelet releasing system further includes a raw material container 5, a platelet collecting container 6, a driving liquid container 7 and a pressure control container 8, the raw material container 5 is connected to the raw material inlet 1011 through a first pipe 9, the platelet collecting container 6 is connected to the product outlet 1012 through a second pipe 10, the driving liquid container 7 is connected to the driving liquid inlet 1013 through a third pipe 11, and the pressure control container 8 is connected to the pressure control port 1014 through a fourth pipe 12. Specifically, luer female ports 15 are provided at the respective through ports, and luer fittings (not shown) are used as connectors to connect the respective containers. A pressure adjusting device 13 and a flow adjusting device 14 connected to the raw material container 5, a pressure adjusting device 13 and a flow adjusting device 14 connected to the platelet collecting container 6, and a flow adjusting device 14 connected to the fourth tube 12. In the present embodiment, the pressure regulator 13 is an electric pressure regulating valve, and the flow regulator 14 is a gas-liquid solenoid valve.
Example 2
The present embodiment provides a method for producing platelets by using the platelet releasing system shown in fig. 9 of embodiment 1, which is shown in fig. 10, and specifically includes:
pluripotent stem cell-induced megakaryocytes (purchased from thermopdissher) in an IMDM/F12 (3. And increasing the pressure of the upper half cavity to 10 mbar-100 mbar to extrude cells, wherein the cell membranes can be broken by too strong pressure to cause cell rupture, and the cell bodies cannot be effectively extruded by too weak pressure to be beneficial to the generation of platelets by the action of shearing force on the cell bodies. Then, a pressure of 100-400mbar is applied to the lower half cavity part 102b, a culture medium of IMDM/F12 (3. 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 strong, and the platelet is activated and the platelet activity is changed when the shearing force is too large; the flow rate is too weak, the platelet production efficiency is lowered, and finally the platelets 17 are collected.
Microscopic observation is carried out on the process, wherein fig. 11 is the residual situation of the megakaryocytes after the megakaryocytes pass through the filter pores, and the residual situation of the megakaryocytes in the picture shows that the megakaryocytes are smoothly captured to the filter pores; FIG. 12 is a fluorescence labeling diagram of cytoskeleton after the sub-membrane megakaryocytes are deformed under force, wherein 12a is the shape of the megakaryocytes before deformation, and 12b is the deformation condition of the megakaryocytes after the megakaryocytes are extruded and deformed and are acted by shearing force, and the deformation condition of the megakaryocytes in the diagram shows that the megakaryocytes are extruded at the filter holes; FIG. 13 is a fluorescent labeling of recovered platelets (CD 41 +) with results showing sizes of 2-4 microns, consistent with platelet size, produced by megakaryocytes in filter wells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

1. An in vitro platelet delivery system comprising:
the micro-fluidic chip 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 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 holes are conical, and the large openings of the filter holes face the upper half cavity part;
a pressure regulating device that can respectively regulate the pressure of the upper half chamber portion and the lower half chamber portion;
and
and the flow regulating device can respectively regulate the liquid flow entering the upper half cavity part and the liquid flow entering the lower half cavity part.
2. The system for releasing platelets in vitro according to claim 1, wherein the lower half cavity portion is internally provided with a turbulence member, and the turbulence member comprises a turbulence member body and a plurality of obstacles distributed on the turbulence member body.
3. A platelet extracorporeal release system according to claim 2, wherein the barrier is a planar directional projection comprising a long arm and a short arm, the long arm and the short arm forming an included angle of 45 ° to 90 ° with the included angle directed toward the driving liquid inlet.
4. A platelet extracorporeal release system according to claim 1, wherein the lid body further has a pressure control port formed on a surface thereof, the driving liquid inlet and the product outlet communicate with the lower half chamber portion through a first passage, and the raw material inlet and the pressure control port communicate with the upper half chamber portion through a second passage.
5. A platelet in-vitro release system according to claim 1, wherein the cover body comprises an upper cover body and a lower cover body which are mutually matched, the inner parts of the upper cover body and the lower cover body respectively comprise a part of the cavity part, and a soft film layer is arranged between the upper cover body and the lower cover body and is arranged at two sides of the cavity; the soft film layer comprises an upper soft film layer and a lower soft film layer, 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.
6. The in vitro platelet release system of claim 5, 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.
7. The system for releasing platelets in vitro according to claim 4, further comprising 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, and 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 port through a fourth pipeline.
8. A system for extracorporeal platelet release according to claim 1, wherein the filter further comprises a reinforcing ring disposed along a rim of the filter body.
9. A method for producing platelets by using the system for releasing platelets in vitro according to any one of claims 1 to 8, wherein megakaryocytes are introduced into the upper half chamber portion from the raw material inlet, extruded through the filter holes, and then subjected to fluid shearing by introducing a driving liquid from the driving liquid inlet while passing through the filter holes, and finally, the product introduced into the lower half chamber portion is collected.
10. A method of producing platelets according to claim 9, wherein megakaryocytes enter the upper chamber half at a pressure of 5 to 20mbar, fluid in the lower chamber half through the filter holes has a pressure of 10 to 100mbar, and media is driven into the lower chamber half at a pressure of 100 to 500mbar at a flow rate of 5 to 15ml/min.
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