Temperature-sensitive PTFE membrane dynamic co-culture device and cell directional separation method thereof

Abstract

The invention discloses a temperature-sensitive PTFE membrane dynamic co-culture device and a cell directional separation method thereof in the technical field of biomechanical engineering research, a double-layer culture dish of a polytetrafluoroethylene composite membrane with the aperture of 10-50 microns is embedded in the middle of the device, poly (N-isopropylacrylamide) temperature-sensitive hydrogel is grafted on the surface of the membrane, the temperature-sensitive hydrogel is hydrophilic at the temperature of 37 DEG C to promote cell adhesion, and the cell directional separation effect is achieved. The hydrophobic trigger cell exfoliation is carried out below 25 DEG C; the motor drives the culture dish to rotate, the upper and lower Peltier modules independently control the temperature, magnetic sealing realizes rapid layering, and membrane holes allow local cells and signal molecules to pass through to intercept the body. The device solves the problems of cell mixing, separation damage and insufficient dynamic simulation in traditional co-culture, supports direct co-culture of at most three cells, is suitable for physiological and pathological studies of nerve cells, tumor cells and the like, and provides an efficient tool for in-vitro cell interaction analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomechanical engineering research, and in particular to a temperature-sensitive PTFE membrane dynamic co-culture device and a cell directional separation method thereof. Background Art

[0002] In biomedical research, cell co-culture technology provides a key platform for studying intercellular signal transmission, functional regulation and pathological mechanisms by simulating the in vivo cell microenvironment. The existing technology mainly realizes cell co-culture through porous membrane separation. For example, the Transwell indirect co-culture system uses a static polycarbonate membrane to allow signal molecules to diffuse, but cannot support direct contact of local cells (such as nerve axons and tumor cell pseudopodia); although the microfluidic chip can simulate fluid shear force, its structure is complex, the cost is high and it is difficult to be compatible with traditional culture vessels. Chinese patent CN101942389A discloses a mechanical loading flow chamber device that applies shear stress to cells on both sides of the membrane through a dual flow channel, but the fixed flow channel design limits the dynamic regulation of the cell contact mode, and the polycarbonate membrane has insufficient mechanical strength. Long-term culture is prone to deformation and causes cell mixing. Another patent CN209685813U uses a magnetic polycarbonate membrane to separate the culture dish. Although it achieves preliminary separation of cells, the biocompatibility of the membrane material is limited, and the reliance on mechanical disassembly leads to a high cell damage rate, which cannot meet the needs of high-precision cell separation.

[0003] However, these technologies generally suffer from three core flaws: First, static membrane structures cannot simulate the dynamic microenvironment in the body; cells interact only indirectly through diffusion signals, resulting in low branch contact efficiency, making it difficult to reproduce physiological processes such as the directional growth of nerve cell axons and the three-dimensional interaction between tumor cells and stromal cells. Second, membrane material performance is limited. Traditional polycarbonate and polyethersulfone membranes lack temperature-sensitive responsiveness, and adhesion regulation relies on chemical reagents (such as pancreatic enzymes), which easily damage cell surface proteins. Third, cell separation technology is backward. Mechanical scraping or chemical digestion leads to low cell survival rates (often below 85%), and there is a lack of a precise separation mechanism that coordinates pore size and temperature sensitivity. Cross-contamination rates are generally higher than 5%. With the deepening of research in neuroregenerative medicine and tumor microenvironment, existing technologies can no longer meet the urgent needs for dynamic cell co-culture and non-destructive separation. There is an urgent need to develop new devices that combine environmental simulation capabilities with intelligent separation functions. Summary of the Invention

[0004] The present invention aims to provide a temperature-sensitive PTFE membrane dynamic co-culture device and a cell directional separation method thereof to solve the problem that cells cannot be directly co-cultured, normal cell signal conduction cannot be achieved, and the two types of cells cannot be separated.

[0005] To solve the above technical problems, the present invention provides the following technical solutions: a temperature-sensitive PTFE membrane dynamic co-culture device, comprising a box body, a culture dish and a polytetrafluoroethylene composite membrane, wherein a motor is connected to the inner wall of one side of the box body, and the output shaft of the motor passes through the inner wall of the box body and is connected to the side wall of the culture dish. The pore size of the polytetrafluoroethylene composite membrane is 10-50 μm, and the culture dish is divided into an upper chamber and a lower chamber by the polytetrafluoroethylene composite membrane. The upper chamber and the lower chamber are both connected to a Peltier temperature control module, and the bottom end of the upper chamber is magnetically connected to the top end of the lower chamber. The surface of the polytetrafluoroethylene composite membrane is grafted with poly (N-isopropylacrylamide) temperature-sensitive hydrogel, and the hydrophilic contact angle at 37°C is less than 30°, and the hydrophobic contact angle at 25°C is greater than 100°.

[0006] Furthermore, the polytetrafluoroethylene composite membrane is formed by compounding expanded polytetrafluoroethylene and carbon fibers with a diameter of 50-200 nm, has a tensile strength of ≥50 MPa, and has a surface fluorination treatment to enhance wear resistance.

[0007] Furthermore, the poly N-isopropylacrylamide thermosensitive hydrogel layer has a thickness of 1-5 μm and is grafted onto the surface of the PTFE membrane by plasma treatment or surface-initiated atom transfer radical polymerization, with a grafting density of 0.1-1.0 mg / cm 2 .

[0008] Furthermore, the edge of the polytetrafluoroethylene composite membrane is sealed and connected to the inner wall of the culture dish via an elastic silicone sealing ring.

[0009] Furthermore, the method for directional cell separation according to any of the above devices comprises the following steps:

[0010] S1. Co-culture stage: Cells A are inoculated on the upper layer of the culture dish at 37°C. After cells A adhere to the wall, the culture dish is turned over and cells B are inoculated on the lower layer. Sufficient culture medium is added to submerge the polytetrafluoroethylene composite membrane, so that cells A and cells B adhere to the upper and lower surfaces of the polytetrafluoroethylene composite membrane, respectively. After cells B adhere to the wall, the culture medium is replaced and suspended cells C are added. After the addition of suspended cells C, the culture dish is rotated 90° to stand upright to prevent suspended cells C from adhering to the wall, and culture medium is added.

[0011] S2, dynamic culture: start the motor to rotate the culture dish, and maintain the culture temperature through the Peltier module to simulate the temperature environment in the body;

[0012] S3. Directional separation: First extract the suspended cells C, restore the culture dish to a horizontal state, absorb all the culture medium and then centrifuge to obtain the suspended cells C; start the motor to rotate the culture dish to keep it horizontal, then lower the temperature to below 25°C to hydrophobize the surface of the polytetrafluoroethylene composite membrane. After collecting the lower layer of cells B, repeatedly rinse the culture chamber, rotate the culture dish 180°, and collect cells A in the same way.

[0013] Furthermore, in step S3, the surface adhesion of the polytetrafluoroethylene composite membrane is increased from 10-15 dyn / cm in the culture state to 2 Reduced to <5dyn / cm 2 , cell survival rate>95%, cross contamination rate<1%.

[0014] The beneficial effects of the present invention are as follows: 1. The motor drives the culture dish to rotate and combines with the Peltier module for independent temperature control, which can simulate the fluid shear force and temperature microenvironment in the body, support local cells (such as nerve axons and tumor cell pseudopodia) to directly contact through the 10-50μm pore size PTFE membrane, significantly improve the efficiency of intercellular signal transmission and physical interaction, and solve the problem of insufficient dynamic simulation of traditional static membranes.

[0015] 2. The thermosensitive poly (N-isopropylacrylamide) hydrogel grafted onto the membrane surface is hydrophilic at 37°C, promoting cell adhesion (contact angle <30°), and hydrophobic below 25°C, triggering natural cell detachment (contact angle >100°). No chemical reagents (such as trypsin) are required, with a cell survival rate >95% and a cross-contamination rate <1%, overcoming the cell damage and high contamination problems caused by traditional separation technologies.

[0016] 3. The double-layer culture dish is quickly separated by magnetic sealing and supports direct co-culture of up to three cell types (two adherent and one suspended). The membrane pores intercept the cell body and allow signal molecules and branches to pass through, taking into account both material exchange and independent cell culture. It is suitable for physiological and pathological studies of nerve cells, tumor cells, etc.

[0017] 4. The PTFE composite membrane (composite of expanded polytetrafluoroethylene and carbon fiber) has a tensile strength of ≥50 MPa, and its surface fluorination treatment enhances wear resistance. It can be reused ≥10 times. It has a rapid temperature-sensitive response (cell detachment is completed within 10 minutes), and separation time is less than 5 minutes, significantly improving experimental efficiency and providing an efficient, non-destructive, and universal tool for in vitro cell interaction analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic structural diagram of a temperature-sensitive PTFE membrane dynamic co-culture device of the present invention. DETAILED DESCRIPTION

[0019] The following is further described in detail through specific implementation methods:

[0020] The reference numerals in the drawings of the specification include: box body 1, motor 101, magnet 2, upper chamber 3, Peltier temperature control module 4, connecting column 5, polytetrafluoroethylene composite film 6, bearing 7.

[0021] The embodiment is basically as shown in the attached Figure 1As shown: A temperature-sensitive PTFE membrane dynamic co-culture device, including a box body 1, a culture dish and a polytetrafluoroethylene composite membrane 6, a motor 101 is fixedly connected to the inner wall of one side of the box body 1, the output shaft of the motor 101 passes through the inner wall of the box body 1 and is connected to a micro bearing 7, the micro bearing 7 is fixedly connected to the side wall of the culture dish, the outer ring of the micro bearing 7 is provided with anti-slip grooves, the pore size of the polytetrafluoroethylene composite membrane 6 is 10-50μm, the polytetrafluoroethylene composite membrane 6 is composed of expanded polytetrafluoroethylene and a diameter of 50-200nm The surface of the polytetrafluoroethylene composite membrane is grafted with poly (N-isopropylacrylamide) thermosensitive hydrogel, with a hydrophilic contact angle of <30° at 37°C and a hydrophobic contact angle of >100° at 25°C. The thickness of the poly (N-isopropylacrylamide) thermosensitive hydrogel layer is 1-5 μm. It is grafted onto the surface of the PTFE membrane by plasma treatment or surface-initiated atom transfer radical polymerization, with a grafting density of 0.1-1.0 mg / cm 2 The culture dish is divided into an upper chamber 3 and a lower chamber by a polytetrafluoroethylene composite membrane 6. The top of the upper chamber 3 is slidably connected to a movable cover. The edge of the polytetrafluoroethylene composite membrane 6 is sealed with the upper chamber 3 and the lower chamber through an elastic silicone sealing ring. A Peltier temperature control module 4 is fixedly connected to the upper chamber 3 and the lower chamber. The bottom end of the upper chamber 3 is magnetically connected to the top end of the lower chamber by a magnet 2.

[0022] A method for directional cell separation using a temperature-sensitive PTFE membrane dynamic co-culture device comprises the following steps:

[0023] S1, co-culture stage: two types of cells were inoculated in the upper and lower layers of the culture dish at 37°C, and the cells adhered to the surface of the polytetrafluoroethylene composite membrane, with the branches contacting through the membrane pores;

[0024] S2, dynamic culture: Start the motor to rotate the culture dish, and maintain the culture temperature through the Peltier module to simulate the fluid shear force and temperature environment in the body;

[0025] S3, directional separation: first extract the suspended cells C, restore the culture dish to a horizontal state, absorb all the culture medium and then centrifuge to obtain the suspended cells C; start the motor to rotate the culture dish to keep the culture dish horizontal, then reduce the temperature to below 25 ° C, the surface of the polytetrafluoroethylene composite membrane is hydrophobic, collect the lower layer of cells B, repeatedly rinse the culture chamber, rotate the culture dish 180 degrees, and collect cells A in the same way. When the cells fall off, the adhesion force of the polytetrafluoroethylene composite membrane surface changes from 10-15 dyn / cm in the culture state to 10-15 dyn / cm 2 Reduced to <5dyn / cm 2 , cell survival rate>95%, cross contamination rate<1%.

[0026] The specific implementation process is as follows:

[0027] 1. Experimental preparation stage

[0028] Device Sterilization and Performance Validation

[0029] After high-pressure sterilization, the sealing performance was tested by the methylene blue leakage method. The results showed that after standing for 24 hours, there was no visible leakage at the edge of the membrane (leakage rate <0.01%, Table 1). The combination of the elastic silicone sealing ring and the magnetic seal met the biosafety-level sealing requirements.

[0030] Scanning electron microscopy (SEM) observations of the pore size distribution of the PTFE membrane showed an average pore size of 5.2±0.3 μm (CV=5.8%, Table 1), which is highly consistent with the design target (5 μm), ensuring that cells with a diameter greater than 20 μm cannot pass through, while local cells can pass freely.

[0031] Table 1 Performance parameters of key components of the device

[0032] Test items Experimental methods Measured data Design indicators Membrane pore size SEM image analysis 5.2±0.3μm (n=50 fields of view) 5±1μm Seal leakage rate Methylene blue permeabilization method <0.01% (24h observation) ≤0.1％ Contact angle of thermosensitive film (37°C) Contact angle meter 25±2°(n=10 tests) ＜30° Contact angle of thermosensitive film (20℃) Contact angle meter 125±3°(n=10 tests) ＞100°

[0033] 2. Cell Seeding and Initial Culture

[0034] Adherent cell A seeding (upper layer)

[0035] 5×10 4 cells / cm 2 Adherent cells A were seeded at a high density, and the attachment rate reached 95.3±1.2% after 2 hours (trypan blue staining count, n=3), proving that the hydrophilicity of the poly (N-isopropylacrylamide) (PNIPAAm) thermosensitive membrane significantly improved the cell adhesion efficiency at 37°C (Table 2).

[0036] Adherent cell B seeding (lower layer)

[0037] After adherent cells A adhered to the membrane, the culture dish was flipped over and adherent cells B were inoculated on the lower layer. Observation under an inverted microscope 24 hours after inoculation showed that adherent cells B were attached to the lower surface of the membrane, with a cell protrusion extension rate of 76.5±3.2% and an average protrusion length of 60±10 μm (n=100 cells, Table 2). Among them, 58.3% of the protrusions grew through the membrane pores to the upper layer, forming initial contact sites.

[0038] Table 2 Comparison of cell adhesion and neurite extension performance

[0039] Cell type Adhesion rate (2h) Protrusion elongation (24h) Average protrusion length (μm) Adherent cell A 95.3±1.2％ 81.3±4.5％ 50±8 Adherent cells B 98.2±0.8％ 76.5±3.2％ 60±10 Traditional membrane 82.7±2.1％ 55.6±5.0％ 40±12

[0040] 3. Dynamic Co-cultivation Stage

[0041] Conventional rotary culture (37°C, 2 rpm)

[0042] After 48 hours of culture, immunofluorescence staining showed that the efficiency of axons / processes of adherent cells passing through the membrane pores was significantly improved compared with static culture. Fluid dynamics calculations showed that the shear force on the membrane surface was 0.6±0.1dyn / cm at a rotation speed of 2rpm. 2 , which is highly matched with the cell microenvironment in vivo.

[0043] Verification of culture medium sharing characteristics

[0044] By fluorescently labeling cytokines (such as IL-6), it was confirmed that the uniformity of fluorescence signal intensity in the upper and lower culture media was >98%, indicating that the cytokines secreted by adherent cells were evenly distributed in the entire culture system without regional concentration differences.

[0045] Table 3 Effects of dynamic culture on local cell growth

[0046]

[0047] IV. Addition and Culture of Suspension Cells

[0048] Suspension cell C seeding

[0049] After adherent cells A and B are completely attached to the wall, the culture medium is replaced and suspended cells C are added. The culture dish is then rotated 90° and placed upright. Dynamic rotation (0.1-10 rpm) is used to maintain the suspended state of suspended cells C to prevent them from adhering to the wall of the culture dish.

[0050] 5. Cell Directed Separation Stage

[0051] Temperature-sensitive triggering and magnetic separation effect

[0052] After cooling to 20℃, the cell detachment dynamics showed that more than 90% of the adherent cells were detached within 10 minutes, and the membrane surface adhesion strength increased from 12.3±1.5dyn / cm 2 Dropped to 3.6±0.8dyn / cm 2 (Real-time monitoring by mechanical sensors).

[0053] Separation sequence optimization

[0054] Collection of suspended cells C: Return the culture dish to a horizontal state, aspirate all the culture medium, and then centrifuge (1000 rpm, 5 minutes) to obtain suspended cells C.

[0055] Separation of adherent cells: The upper chamber was removed by magnetic attraction and the suspensions of adherent cells A and B were collected separately. The cross-contamination rate was <1% as determined by flow cytometry.

[0056] Comparison of cell survival rate

[0057] Trypan blue exclusion test showed that the survival rate of adherent cells after separation was 96.1±1.2%, and the survival rate of suspension cells was 95.8±1.0%, which were significantly higher than those of traditional chemical digestion method (p<0.001, Table 4), confirming the advantage of separation without chemical damage.

[0058] Table 4 Performance comparison of different separation methods

[0059] index This device Transwell mechanical separation Pancreatic digestion Cross contamination rate ＜1％ 5.2±1.1％ 4.8±0.9％ Adherent cell survival rate 96.1±1.2％ 85.2±2.0％ 82.7±2.0％ Suspension cell viability 95.8±1.0％ — — Separation time ≤ 5 minutes >30 minutes >15 minutes

[0060] 6. Long-term stability and functional verification

[0061] Membrane material durability test

[0062] After 14 days of continuous culture, the tensile strength of the PTFE composite membrane decreased from 55 MPa to 49.5 MPa (retention rate 90%), and the surface wear rate was less than 5% (roughness Ra increased from 0.2 μm to 0.23 μm, Table 5), proving that the material can be reused ≥10 times (the measured temperature-sensitive conversion efficiency is still >90% after 10 cycles).

[0063] Table 5 Long-term performance and functional verification of the device

[0064]

[0065] This example systematically verifies the thermosensitive PTFE membrane dynamic co-culture device and the cell directional separation method. Through material performance testing, multi-cell co-culture process optimization and separation efficiency evaluation, the technical advantages and practicality of the device are fully demonstrated. The core component of the device, the polytetrafluoroethylene (PTFE) composite membrane, has a pore size of 5.2±0.3μm and is grafted with poly N-isopropylacrylamide (PNIPAAm) thermosensitive hydrogel on the surface. At 37°C, the hydrophilic contact angle of 25±2° promotes cell adhesion, and at 20°C, the hydrophobic contact angle of 125±3° triggers cell detachment. The adhesion force increases from 12.3±1.5dyn / cm 2 Dropped to 3.6±0.8dyn / cm 2 Combining a magnetic seal with an independent temperature control module, the device achieves dynamic microenvironment simulation and damage-free separation. Experiments have shown that the device's seal leakage rate is less than 0.01%, the membrane material's tensile strength retention rate is 90% (after 14 days), and it can be reused 10 times or more, meeting long-term culture needs.

[0066] During the cell culture stage, the adherence rate of adherent cells exceeded 95% at 37°C for 2 hours, the protrusion extension rate reached more than 75% after 24 hours, and 58.3% of the protrusions passed through the membrane pores to form transmembrane contacts, which was about 40% higher than that of traditional membranes; verified by fluorescent labeling, the uniformity of cytokines in the upper and lower culture media was >98%, ensuring uniform distribution of signal molecules. After adding suspended cells, the culture dish was rotated 90° upright and dynamically rotated to effectively maintain the suspension state and avoid adhesion. During separation, the suspended cells were first collected by centrifugation (survival rate 95.8±1.0%), and then cooled to below 25°C to allow the adherent cells to fall off naturally. After magnetic stratification, the cross-contamination rate was <1%, and the cell survival rate was >96%, which was significantly better than Transwell mechanical separation (cross-contamination rate 5.2%) and trypsin digestion (survival rate <85%), and the separation time was shortened to less than 5 minutes.

[0067] Through material modification (PTFE composite membrane enhances mechanical properties), structural innovation (flippable upright culture dish), and methodological breakthroughs (temperature-sensitive triggered separation), the device has established a "dynamic co-culture-intelligent separation" technology system. It supports the direct co-culture of up to three cell types (two adherent and one suspended), addressing the problems of cell mixing, separation damage, and insufficient dynamic simulation in traditional technologies. Measured data demonstrates significant advantages in the study of the physiology and pathology of neural cells, tumor cells, and other cells. It provides an efficient and versatile tool platform for in vitro cell interaction analysis, combining scientific and engineering practicality.

[0068] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A temperature-sensitive PTFE membrane dynamic co-culture device, characterized by: The device comprises a box body, a culture dish and a polytetrafluoroethylene composite membrane. A motor is connected to the inner wall of one side of the box body, and the output shaft of the motor passes through the inner wall of the box body and is connected to the side wall of the culture dish. The pore size of the polytetrafluoroethylene composite membrane is 10-50 μm. The culture dish is divided into an upper chamber and a lower chamber by the polytetrafluoroethylene composite membrane. Peltier temperature control modules are connected to the upper chamber and the lower chamber. The bottom end of the upper chamber is magnetically connected to the top end of the lower chamber. The surface of the polytetrafluoroethylene composite membrane is grafted with poly (N-isopropylacrylamide) thermosensitive hydrogel, and the hydrophilic contact angle is less than 30° at 37°C and the hydrophobic contact angle is greater than 100° at 25°C.

2. A temperature-sensitive PTFE membrane dynamic co-culture device according to claim 1, characterized in that: The polytetrafluoroethylene composite membrane is formed by compounding expanded polytetrafluoroethylene and carbon fibers with a diameter of 50-200 nm, has a tensile strength of ≥50 MPa, and has a surface fluorination treatment to enhance wear resistance.

3. A temperature-sensitive PTFE membrane dynamic co-culture device according to claim 2, characterized in that: The poly (N-isopropylacrylamide) thermosensitive hydrogel layer has a thickness of 1-5 μm and is grafted onto the surface of the PTFE membrane by plasma treatment or surface-initiated atom transfer radical polymerization, with a grafting density of 0.1-1.0 mg / cm 2 .

4. A temperature-sensitive PTFE membrane dynamic co-culture device according to claim 3, characterized in that: The middle part of the side wall of the culture dish is connected to the output shaft of the driving motor through a micro bearing, and the outer ring of the micro bearing is provided with anti-slip grooves.

5. A temperature-sensitive PTFE membrane dynamic co-culture device according to claim 4, characterized in that: The edge of the polytetrafluoroethylene composite membrane is sealed and connected to the upper cavity and the lower cavity through an elastic silicone sealing ring.

6. A method for directed cell separation using a device according to any one of the preceding claims, characterized in that: The following steps are involved: S1. Co-culture stage: Cells A are inoculated on the upper layer of the culture dish at 37°C. After cells A adhere to the wall, the culture dish is turned over and cells B are inoculated on the lower layer. Sufficient culture medium is added to submerge the polytetrafluoroethylene composite membrane, so that cells A and cells B adhere to the upper and lower surfaces of the polytetrafluoroethylene composite membrane, respectively. After cells B adhere to the wall, the culture medium is replaced and suspended cells C are added. After the addition of suspended cells C, the culture dish is rotated 90° to stand upright to prevent suspended cells C from adhering to the wall, and culture medium is added. S2, dynamic culture: start the motor to rotate the culture dish, and maintain the culture temperature through the Peltier module to simulate the temperature environment in the body; S3, Directional separation: First extract the suspended cells C, restore the culture dish to a horizontal state, aspirate all the culture medium and then centrifuge to obtain the suspended cells C; Start the motor to rotate the culture dish and keep it horizontal. Then lower the temperature to below 25°C to make the surface of the polytetrafluoroethylene composite membrane hydrophobic. After collecting the lower layer of cells B, repeatedly rinse the culture chamber, rotate the culture dish 180°, and collect cells A in the same way.

7. The method for directional cell separation using a temperature-sensitive PTFE membrane dynamic co-culture device according to claim 6, characterized in that: In step S3, the surface adhesion of the polytetrafluoroethylene composite membrane is increased from 10-15 dyn / cm in the culture state. 2 Reduced to <5dyn / cm 2 , cell survival rate>95%, cross contamination rate<1%.

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