Uniaxial adjustable mechanical stretching device for organoids or cells and stretching culture method

Abstract

The invention discloses a single-shaft adjustable mechanical stretching device for organoids or cells and a stretching culture method. A fixed support and a guide rail are arranged on a fixed base through a guide rail lifting seat, and a sliding block above the guide rail is connected with a stretching movement end through an adapter seat; the stretching moving end and the stretching fixed end are arranged at an interval, a lead screw of a stepping motor on the fixed support is fixedly connected to a coupler, and the coupler is fixedly connected with an adapter through a blocking block; handles on the two sides of the culture chamber are fixed to a stretching fixing base and a stretching fixing end respectively, a stepping motor is started, a lead screw of the stepping motor drives an adapting base to move longitudinally when moving, the longitudinal movement of the adapting base drives a sliding block to move on a guide rail, and then a stretching movement end is made to be far away from / close to the corresponding stretching fixing end; according to the invention, accurate and controllable tensile mechanical stimulation is carried out on different forms of organoids or cells through the replaceable culture chamber and the accurately controllable stepping motor, so that the tissue maturity / calcification degree is obviously changed or the cell differentiation process is adjusted.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of organoid tissue engineering and cell culture, and particularly relates to a single-axis adjustable mechanical stretching device for organoids or cells and a stretching culture method. BACKGROUND

[0002] In recent years, organoid culture technology has developed rapidly, providing a powerful platform for in vitro reconstruction of tissue microenvironment, disease modeling, drug screening and regenerative medicine research. However, most existing organoid culture systems still use static culture methods, lacking simulation of physiological mechanical stimulation, resulting in insufficient tissue maturity and single morphological structure. In addition, mechanical force-sensitive heart valve organoids, for example, stress loss can accelerate extracellular matrix component imbalance and affect functional formation.

[0003] The current mainstream organoid culture system still uses static three-dimensional culture, lacking effective simulation of physiological mechanical environment. For example, patent CN112680355B discloses a scalable structure and universal cancer organoid in vitro culture device. Although this method can batch produce organoids, it only focuses on high-throughput production of droplet dot plates and does not integrate any stress loading mechanism. For example, patent CN114107175B discloses an organoid culture method for inducing growth of intestinal organoids by mechanical stretching. This technology can realize stretching of intestinal organoids, but only gives single / dual-axis fixed sinusoidal stretching parameters, lacks specific device examples and real-time monitoring feedback, and has insufficient scalability and multi-model versatility. SUMMARY

[0004] The purpose of the present application is to solve the problems existing in the prior art and provide a single-axis adjustable mechanical stretching device for organoids or cells and a stretching culture method. By replacing the culture chamber and precisely controlling the stepping motor, precise and controllable mechanical stretching stimulation can be achieved on different forms of organoids or cells, thereby significantly changing the tissue maturity / calcification degree or regulating the cell differentiation process.

[0005] Technical scheme: The single-axis adjustable mechanical stretching device for organoids or cells comprises a fixed base, a guide rail, a plurality of stretching fixed ends, a plurality of stretching moving ends, and a plurality of culture chambers.

[0006] The upper surface of the fixed base has a longitudinal guide rail raised seat at each end, and a fixed support is arranged at one end of each guide rail raised seat, and a guide rail is arranged at the other end of each guide rail raised seat. A plurality of sliders are connected to the upper part of the guide rail at intervals, and the sliders are connected to the corresponding stretching moving ends through an adapter seat at the upper part.

[0007] Each stretching fixed seat is uniformly installed between the two guide rails on the upper surface of the fixed base in transverse direction, and the stretching movement end and the stretching fixed end are arranged in pairs with a spacing, and the stretching fixed seat top and the stretching movement end top are both provided with clamping parts; the two sides of the culture chamber are provided with outward protruding handles;

[0008] The fixed support is provided with a stepping motor, a screw rod of the stepping motor is fixedly connected to a shaft coupling, and the shaft coupling is fixedly connected to the adapter seat through a plug block.

[0009] In use, first, the handle on one side of the culture chamber is fixed to the clamping part on the top of the stretching fixed seat, and the handle on the other side is fixed to the clamping part on the top of the stretching fixed end, then the stepping motor is started, so that the screw rod of the stepping motor drives the adapter seat to move longitudinally, the longitudinal movement of the adapter seat drives the sliding block to move along the guide rail, and the stretching movement end is away from / close to the corresponding stretching fixed end.

[0010] The present application can realize precise and controllable stretching mechanical stimulation on different forms of organoids or cells through replaceable culture chambers and precisely controllable stepping motors, thereby significantly changing the maturity / calcification degree of tissues or regulating the differentiation process of cells.

[0011] Further, the lower surface of the fixed base is provided with bosses for anti-skid at four top corners; the clamping parts on the top of the stretching fixed seat and the stretching movement end are detachable through bolts and nuts.

[0012] Further, the stepping motor is connected to a motor driver through wires, the motor driver is connected to a stepping motor controller through wires, and the stroke, proportion, one-way times, cycle times and stretching speed are adjusted through the control screen of the stepping motor controller, so that the stepping motor is precisely controlled to perform cyclic movement, progressive movement and fixed-length movement.

[0013] Further, the culture chamber includes two types: an organoid culture chamber and a cell culture chamber, if it is a valve organoid, the tissue block is fixed to the organoid culture chamber, and if it is an adherent cell, the cell is seeded in the cell culture chamber.

[0014] The present application also discloses a stretching culture method of a single-axis adjustable mechanical stretching device for organoids or cells, comprising the following steps:

[0015] Step 1, sample preparation and inoculation stage;

[0016] The cultured organoid or adherent cell sample is inoculated in the culture chamber, and then the handles on the two sides of the culture chamber are clamped by the stretching fixed end and the stretching movement end;

[0017] Step 2, pre-culture stage;

[0018] Place the assembled single-axis adjustable mechanical stretching device of step 1 in a constant temperature and humidity incubator (for example, the temperature in the incubator is maintained at 37°C and contains 5% CO2) for regular culture for 1-3 days to stabilize the organoid / cell state;

[0019] Step 3, stretching parameter setting stage;

[0020] According to actual needs, set the displacement amplitude, stretching frequency, cycle mode and total duration through the control screen respectively;

[0021] Among them, the displacement amplitude is set to 10% ± 1%, the stretching frequency is set to 1 Hz; the cycle mode refers to the stretching phase and the recovery phase in each single cycle, the stretching phase is set to last for 1s and the recovery phase is set to last for 1s; the total duration is set to cycle 30000 times / day;

[0022] Step 4, implement quantitative stretching stage;

[0023] Start the stepper motor driver, so that the stepper motor drives the lead screw according to the preset program, so that the lead screw of the stepper motor moves to drive the adapter seat to move longitudinally, the longitudinal movement of the adapter seat drives the slider to move along the guide rail, and then the stretching movement end moves away from / approaches the corresponding stretching fixed end, that is, the stretching movement end moves along the guide rail reciprocating, thereby implementing single-axis stretching on the sample in the culture chamber;

[0024] Step 5, sampling and detection: after the culture is completed, stop the device and take out the sample for one or more of the following detections:

[0025] ①Calcification detection: Alizarin Red S staining is used to evaluate the degree of calcium salt deposition in the tissue or cells;

[0026] ②Gene expression detection: qPCR is used to detect the expression of osteogenic marker genes such as Runx2 and Osteocalcin, or differentiation markers such as RUNX2 and α-SMA;

[0027] ③Morphological observation: differences in morphology between the stretching group and the static control group are observed by microscope photography;

[0028] ④Biomechanical detection: the stepper motor applies single-axis load to the organoid until it breaks, the displacement and force relationship curve is recorded by the mechanical sensor, and the stress-strain relationship curve is obtained by normalizing the morphological parameters, and the biomechanical property differences between the stretching group and the control group are analyzed.

[0029] ⑤Biochemical detection: cell proliferation activity (such as CCK-8 method) or protein expression (Western Blot) is detected.

[0030] Advantages: Compared with the prior art, the present application has the following advantages:

[0031] 1. The present application can stretch a large number of organoids: By increasing the number of stretching components on the device (synchronously increasing the stretching movement end and the fixed end, one movement end with one fixed end), batch stretching of organoids can be achieved, ensuring the repeatability of the experiment.

[0032] 2. The present application adopts a modular and replaceable culture chamber design: suitable for various types of organoids and cell models, only the culture chamber needs to be replaced, without the need to design a complete set of equipment for different experiments, reducing the cost of customization;

[0033] 3. The present application can precisely control the stretching parameters: through touch screen input, displacement control with 0.01mm precision, strain amplitude within 5%~20% and frequency control within 0.1Hz~2Hz can be achieved, meeting the needs of various physiological / pathological simulation;

[0034] 4. The present application can inhibit the calcification of valve organoids: experimental results show that after stretching the valve organoids under 10% strain and 1Hz frequency for 9 days, compared with calcification culture, the transmittance decreases by 33.79%, the elastic modulus increases by 72.20kPa, the ultimate tensile strength decreases by 5.22kPa, the yield strength decreases by 2.64kPa, the elongation at break increases by 36.67%, the positive area of Alizarin Red S staining decreases by about 6.4%, the positive area of collagen fibers decreases by 68.56%, the average angle of cell nuclei increases by 15.01°, the average area of cell nuclei increases by 10.86μm 2 , the roundness of cell nuclei increases by 17.08%, the alignment of α-smooth muscle actin increases by 36.25%, and the average length increases by 17.88μm; the parameter changes of calcified valve organoids in the direction of calcification are improved under 10% strain and 1Hz frequency.

[0035] 5. The present application has wide application: can be applied to various tissue / cell in vitro mechanical researches such as valve organoids, intestinal organoids, myocardial organoids, neural organoids, muscle cells, as well as drug screening and regenerative medicine research. DETAILED DESCRIPTION

[0036] Figure 1 is a structural schematic diagram of the present application;

[0037] Figure 2 is a structural top view of the present application;

[0038] Figure 3 is a structural front view of the present application;

[0039] Figure 4 is a structural side view of the present application;

[0040] Figure 5 Schematic diagram of replaceable organoid stretching culture chamber;

[0041] Figure 6 Top view of replaceable organoid stretching culture chamber;

[0042] Figure 7 Front view of replaceable organoid stretching culture chamber;

[0043] Figure 8 Side view of replaceable organoid stretching culture chamber;

[0044] Figure 9 Schematic diagram of replaceable cell stretching culture chamber;

[0045] Figure 10 Top view of replaceable cell stretching culture chamber;

[0046] Figure 11 Front view of replaceable cell stretching culture chamber;

[0047] Figure 12 Side view of replaceable cell stretching culture chamber;

[0048] Figure 13 Schematic diagram of stretching control screen;

[0049] Figure 14 Comparison diagram of light microscope results after organoid stretching;

[0050] Figure 15 Comparison diagram of biomechanical property parameter results after organoid stretching;

[0051] Figure 16 Comparison diagram of calcium deposition area and collagen deposition area results after organoid stretching;

[0052] Figure 17 Comparison diagram of cell nucleus and α-SMA staining results after organoid stretching.

[0053] Explanation of figure numbers:

[0054] 1, fixed base; 2, stretching fixed end; 3, guide rail lifting seat; 4, guide rail; 5, sliding block; 6, guide rail adapter; 7, stretching moving end; 8, stepping motor; 9, stepping motor fixed support; 10, shaft coupling; 11, plug; 12, organoid stretching culture chamber handle; 13, organoid stretching culture chamber; 14, cell stretching culture chamber handle; 15, cell stretching clamp culture chamber. DETAILED DESCRIPTION

[0055] The technical solutions of the present application will be described in detail below, but the protection scope of the present application is not limited to the described embodiments.

[0056] The present application is based on an alternative culture chamber design, which can implement single-axis precise stretching on any cells or any shape organoids, can cascade expand culture wells to improve the throughput, can set the strain amplitude, frequency and waveform in real time through a touch screen, and reserves multiple sensor interfaces to realize force-strain closed-loop control and real-time imaging feedback, thereby significantly improving the repeatability, flexibility and biological verification depth of the experiment.

[0057] As shown in Figures 1 to 4 A single-axis adjustable mechanical stretching device for organoids or cells according to the present application comprises a fixed base 1, guide rails 4, multiple stretching fixed ends 2, multiple stretching moving ends 7 and multiple culture chambers. The upper surfaces of the fixed base 1 are provided with two longitudinal guide rails 4 at both ends, each of which is provided with a fixed support 9 at one end and a guide rail 4 at the other end. The guide rails 4 are connected to multiple sliders 5 at intervals above the guide rails 4, and the sliders 5 are connected to corresponding stretching moving ends 7 through an adapter seat 6 above the sliders 5. Each stretching fixed end is uniformly installed horizontally between the two guide rails 4 on the upper surface of the fixed base 1, and the stretching moving ends 7 and the stretching fixed ends 2 are arranged at intervals. The top of the stretching fixed end and the top of the stretching moving end 7 are provided with clamping parts. The culture chambers are provided with outwardly protruding handles on both sides. The fixed support 9 is provided with a stepping motor 8, the lead screw of which is fixed to a coupling 10, and the coupling 10 is fixed to the adapter seat 6 through a plug 11. In use, first fix one handle of the culture chamber to the clamping part on the top of the stretching fixed end, and fix the other handle to the clamping part on the top of the stretching fixed end 2, then start the stepping motor 8, so that the lead screw of the stepping motor 8 drives the adapter seat 6 to move longitudinally, and the longitudinal movement of the adapter seat 6 drives the slider 5 to move along the guide rail 4 (i.e. the slider 5 can move linearly relative to the guide rail 4), thereby making the stretching moving end 7 move away from / close to the corresponding stretching fixed end 2.

[0058] The four top corners of the lower surface of the fixed base 1 are provided with bosses for lifting the device to leave space. The clamping parts on the top of the stretching fixed end and the top of the stretching moving end 7 are detachable through bolts and nuts.

[0059] The step motor 8 of the embodiment is connected with the motor driver through wires, the motor driver is connected with the step motor 8 controller through wires, the stroke, the proportion, the one-way frequency, the cycle frequency and the stretching speed are adjusted through the control screen of the step motor 8 controller, and the step motor 8 is accurately controlled to perform the cycle movement, the progressive movement and the fixed-length movement. The step motor 8 adopts a 42 through-type screw rod linear step motor 8, the body height is 48 mm, the rated torque is 52 N·cm, the step angle is 1.8°, the screw rod diameter is 8 mm, the lead is 2 mm, and the minimum displacement resolution is 0.01 mm; the step motor 8 is installed on the step motor 8 fixer and connected with the guide rail 4 adapter component through the shaft coupling 10, the step drive leads the screw rod, and drives the movement end to perform linear movement. The driver type and the control screen of the step motor 8 can be the existing technology, for example, MT542-AL, MTSO35WO2RGD, and the operation interface of the control screen includes a stretching parameter setting page and a fault alarm page. If the force value needs to be fed back, a small force sensor (such as a range of 10 N) can be installed between the movement end and the clamp, as shown in Figure 13 .

[0060] The culture chamber of the embodiment includes two types: an organoid culture chamber and a cell culture chamber. If the valve organoid is used, the tissue block is fixed in the organoid culture chamber, as shown in Figures 5 to 8 , and if the adherent cell is used, the cell is seeded in the cell culture chamber, as shown in Figures 9 to 12 . During the culture, the adherent cell is directly inoculated into the culture chamber, and the culture chamber is fixed on the movement end and the fixed end by tightening the nut.

[0061] The application further discloses a stretching culture method of the single-axis adjustable mechanical stretching device for the organoid or the cell, and the method comprises the following steps.

[0062] Step 1, sample preparation and inoculation stage

[0063] The inoculated organoid or adherent cell sample is inoculated into the culture chamber, and the handles on both sides of the culture chamber are clamped by the stretching fixed end 2 and the stretching movement end 7;

[0064] Step 2, pre-culture stage

[0065] The single-axis adjustable mechanical stretching device assembled in step 1 is placed in a constant-temperature and constant-humidity incubator (37℃, 5% CO2), and conventional culture is performed for 1-3 days, so that the state of the organoid / cell is stable;

[0066] Step 3, stretching parameter setting stage

[0067] The displacement amplitude, the stretching frequency, the cycle mode and the total duration are respectively set through the control screen according to actual requirements;

[0068] Wherein, the displacement amplitude is set to 10% ± 1%, and the stretching frequency is set to 1 Hz; the cycle mode refers to the stretching phase and the recovery phase in each single cycle, the stretching phase is set to last for 1 s, and the recovery phase is set to last for 1 s; the total duration is set to 30000 cycles / day;

[0069] Step 4, implement quantitative stretching phase;

[0070] Start the stepper motor 8 driver, so that the stepper motor 8 drives the lead screw according to the preset program, so that the lead screw of the stepper motor 8 drives the adapter seat 6 to move longitudinally, the longitudinal movement of the adapter seat 6 drives the sliding block 5 to move along the guide rail 4, and then the stretching movement end 7 moves away from / approaches the corresponding stretching fixed end 2, that is, the stretching movement end 7 reciprocates along the guide rail 4, so as to implement uniaxial stretching on the sample in the culture chamber;

[0071] Step 5, sampling and detection: after the culture is completed, stop the device, and take out the sample to perform one or more of the following detections:

[0072] ①Calcification detection: Alizarin Red S staining is used to evaluate the degree of calcium salt deposition of the tissue or cells;

[0073] ②Gene expression detection: qPCR is used to detect the expression of osteogenesis marker genes such as Runx2 and Osteocalcin, or differentiation markers such as RUNX2 and α-SMA;

[0074] ③Morphological observation: differences in morphology between the stretching group and the static control group are compared by taking pictures under a microscope;

[0075] ④Biomechanical detection: the stepper motor 8 applies uniaxial load to the organoid until it breaks, the displacement-force relationship curve is recorded by the mechanical sensor, and the stress-strain relationship curve is obtained by normalizing the morphological parameters, and the differences in biomechanical properties between the stretching group and the control group are analyzed.

[0076] ⑤Biochemical detection: cell proliferation activity (such as CCK-8 method) or protein expression (Western Blot) is detected.

[0077] The embodiment performs in vitro stretching culture on valve organoids, and the specific process is as follows:

[0078] Step 1, mix A and B of 5° food-grade liquid mold silicone (Zhongheng 1105) in equal proportions, stir uniformly, then use a vacuum pump to extract air bubbles, and place the stretching culture chamber and its cover in an oven for more than 2 h;

[0079] Step 2, stretch the organoids culture chamber, organoids stretch culture chamber cover, insect needle, PDMS mold in 75% ethanol solution, ultrasonic cleaning 30 min, then stretch the chamber and stretch the chamber cover, insect needle, PDMS mold, nylon paper frame wrapped in tin paper, in the sterilization pot high temperature and high pressure sterilization. After sterilization, the stretch chamber is sprayed with alcohol, and the ultraviolet is irradiated for 30 min in the transfer window, and then it is taken into the cell room for standby;

[0080] Step 3, put the stretch device main body and the organoids stretch culture chamber, paper frame, PDMS mold and insect needle into the transfer window, spray alcohol, and irradiate ultraviolet for 30 min, then take it into the cell room, and then tighten the stretch device main body and the organoids stretch culture chamber with nut, and irradiate ultraviolet for 8 h for standby;

[0081] Step 4, wash the PDMS mold with sterile water twice, then incubate the mold with 4‰ F127 overnight. Wash the F127 with sterile water, and then fix the paper frame on the PDMS mold with the insect needle;

[0082] Step 5, digest the iPSC-VICs with collagenase type I (Sigma, C7657) for 30 min, then terminate the collagenase type I digestion with 2 times volume of high-sugar medium, then centrifuge at 1000 rpm for 3 min, and discard the supernatant to obtain the precipitate;

[0083] Step 6, add 1 mL Trypsin-EDTA (0.25%, Gibco) to the precipitate and shake in 37℃ warm water for 3 min, then terminate the digestion with termination medium (high-sugar medium: fetal bovine serum = 1:1), then take 10 μL and count with a blood cell counting board, and centrifuge the cells (1000 rpm, 3 min), discard the supernatant, and obtain the iPSC-VICs from the precipitate;

[0084] Step 7, use the hydrogel system to make strip-shaped valve microtissues, where A liquid: 1X: Thrombin = 58:2.4, B liquid: Fibrinogen: Matrigel: (2X+Colleagen I) = 2:1:2, mix A and B liquids in equal proportions, add 50 μL mixed liquid to each strip-shaped valve microtissue, and make the valve microtissue;

[0085] Step 8, after spotting, place the valve microtissue in a 37℃ incubator for 30 min, then remove the valve microtissue from the PDMS mold. Put it into a 12-well plate, add medium, and place it in a 37℃ incubator for pre-culture for 3 days;

[0086] Step 9, the strip-shaped valve microtissues are taken out from the culture plate and fixed on the specimen needle in the organoid stretching culture chamber, and the stretching program is set: the stretching stroke is 0.8 mm, the cycle stretching is 30000 times / day, the one-way number is 1, and the stretching cycle is 2 s. After starting the system, the stepper motor 8 drives the lead screw according to the set program, so that the moving end assembly drives the stretching moving end to reciprocate, and the fixture relative to the stretching fixed end 2 clamps the valve organoid to implement uniaxial stretching. New culture medium is replaced every two days;

[0087] Step 10, after 9 days, the system is closed, the stepper motor 8 is stopped, the specimen needle is extracted, the valve organoid is gently taken out using a sterile forceps, and then the functional parameter detection is performed.

[0088] 1), light transmittance detection; the organoid bright field picture is taken under 4X bright field, and the light transmittance change is analyzed and recorded.

[0089] 2), biomechanical property detection: the organoid is taken out and fixed on a mechanical sensor, a uniaxial load is applied to the organoid at a quasi-static rate (such as 0.01 mm / s), and the displacement and force curve of the organoid are recorded until the organoid is broken.

[0090] 3), alizarin red staining: the organoid is fixed with 4% paraformaldehyde for 20 min, washed with DPBS for three times, paraffin-embedded, and then paraffin sections are prepared, and then 0.2% Alizarin Red S is used for staining, and the staining area is recorded after neutral resin mounting.

[0091] 4), five-color collagen method staining: the organoid is fixed with 4% paraformaldehyde for 20 min, washed with DPBS for three times, paraffin-embedded, and then paraffin sections are prepared, and then Weigerts hematoxylin is used for staining the cell nucleus, woodstain scarlet-acid fuchsin is used for staining the cytoplasm, resordn-fuchsin is used for staining the elastic tissue; saffron is used for staining the collagen tissue; and alizarin blue is used for staining the matrix (proteoglycan). Finally, the collagen positive area is analyzed after neutral resin mounting.

[0092] 5), immunofluorescence staining: the organoid is fixed with 4% paraformaldehyde for 20 min, washed with DPBS for three times, blocked with 5% donkey serum overnight, incubated with anti-α-SMA primary antibody (1:1000), incubated with fluorescent secondary antibody, and then DAPI is used for staining the nucleus. The fluorescence microscope is observed and photographed.

[0093] The experimental results of the embodiment are as follows:

[0094] 1), compared with the ninth day static calcification group, the light transmittance of the stretching calcification group decreases by 33.79% (see Figure 14 ).

[0095] 2) Compared with the static calcification group on the ninth day, the elastic modulus of the stretch calcification group increased by 72.20 kPa, the ultimate tensile strength decreased by 5.22 kPa, the yield strength decreased by 2.64 kPa, and the elongation at break increased by 36.67% (see Figure 15 ).

[0096] 3) Compared with the static calcification group on the ninth day, the alizarin red positive area of the stretch calcification group decreased by 6.4% (see Figure 16 ).

[0097] 4) Compared with the static calcification group on the ninth day, the collagen fiber positive area of the stretch calcification group decreased by 68.56%, as shown in Figure 16 .

[0098] 5) Compared with the static calcification group on the ninth day, the average nuclear angle of the stretch calcification group increased by 15.01°, the average nuclear area increased by 10.86 μm 2 , and the nuclear roundness increased by 17.08%; the alpha-smooth muscle actin alignment increased by 36.25%, and the average length increased by 17.88 μm (see Figure 17 ).

Claims

1. A uniaxial adjustable mechanical stretching device for organoids or cells, characterized in that: It includes a fixed base, a guide rail, a plurality of stretching fixed ends, a plurality of stretching moving ends and a plurality of culture chambers; A guide rail raising seat is longitudinally provided at each end of the upper surface of the fixed base, a fixed bracket is provided on one end of each guide rail raising seat and a guide rail is provided on the other end of each guide rail raising seat, a plurality of sliders are connected at intervals above the guide rails, and the sliders are connected to the corresponding stretching movement ends through adapter seats; Each stretching fixed base is evenly installed laterally between the two guide rails on the upper surface of the fixed base, and the stretching movement end and the stretching fixed end are spaced apart in pairs. The top of the stretching fixed base and the top of the stretching movement end are both provided with a clamping portion; handles protruding outwards are provided on both sides of the culture chamber; The fixed bracket is provided with a stepper motor, the screw rod of the stepper motor is fixedly connected to the coupling, and the coupling is fixedly connected to the adapter through a blocking block; When in use, first fix the handle on one side of the culture chamber to the clamping part on the top of the stretching fixed seat, and fix the handle on the other side to the clamping part on the top of the stretching fixed end, and then start the stepper motor so that the stepper motor's lead screw moves to drive the adapter to move longitudinally. The longitudinal movement of the adapter drives the slider to move along the guide rail, thereby making the stretching moving end move away from / closer to the corresponding stretching fixed end.

2. The uniaxial adjustable mechanical stretching device for organoids or cells according to claim 1, characterized in that: Bosses are provided at the four top corners of the lower surface of the fixed base; the clamping parts at the top of the stretching fixed seat and the top of the stretching movement end are both disassembled and assembled by bolts and nuts.

3. The uniaxial adjustable mechanical stretching device for organoids or cells according to claim 1, characterized in that: The stepper motor is connected to the motor driver via a wire, and the motor driver is connected to the stepper motor controller via a wire. The stroke, ratio, number of one-way movements, number of cycles, and stretching speed are adjusted through the control screen of the stepper motor controller to accurately control the stepper motor to perform cyclic motion, progressive motion, and fixed-length motion.

4. The uniaxial adjustable mechanical stretching device for organoids or cells according to claim 1, characterized in that: The culture chambers include two types: organoid culture chambers and cell culture chambers. If the cells are valve organoids, the tissue blocks are fixed in the organoid culture chambers. If the cells are adherent cells, the cells are plated in the cell culture chambers.

5. A stretching culture method for organoids or cells according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step 1: Sample preparation and inoculation stage; Inoculate the cultured organoid or adherent cell sample into the culture chamber, and then clamp the handles on both sides of the culture chamber by stretching the fixed end and the stretching moving end; Step 2, pre-cultivation stage; Place the uniaxial adjustable mechanical stretching device assembled in step 1 in a constant temperature and humidity incubator for 1 to 3 days to stabilize the organoid / cell state; Step 3, stretching parameter setting stage; According to actual needs, the displacement amplitude, stretching frequency, cycle mode and total duration can be set through the control screen; The displacement amplitude was set to 10% ± 1%, and the stretching frequency was set to 1 Hz. The cycle mode refers to the stretching phase and the recovery phase in each single cycle. The stretching phase was set to last for 1 s, and the recovery phase was set to last for 1 s. The total duration is set to 30,000 cycles per day; Step 4: Implement quantitative stretching stage; The stepper motor driver is started, so that the stepper motor drives the lead screw according to a preset program, so that the lead screw of the stepper motor drives the adapter to move longitudinally when it moves. The longitudinal movement of the adapter moves the slider along the guide rail, thereby causing the stretching end to move away from or approach the corresponding stretching fixed end, that is, the stretching end to reciprocate along the guide rail, thereby performing uniaxial stretching on the sample in the culture chamber; Step 5: Sampling and testing: After the incubation is complete, stop the device and remove the sample for one or more of the following tests: ① Calcification detection: Alizarin Red S staining is used to assess the degree of calcium salt deposition in tissues or cells; ②Gene expression detection: detection of osteoblast marker gene expression or differentiation markers; ③Morphological observation: Compare the morphological differences between the stretching group and the static control group through microscopic photography; ④ Biomechanical testing: A uniaxial load was applied to the organoids using a stepper motor until they broke. The displacement-force relationship was recorded using a mechanical sensor. The morphological parameters were normalized to form a stress-strain relationship curve to analyze the differences in biomechanical properties between the stretching group and the control group. ⑤Biochemical testing: detecting cell proliferation activity or protein expression.

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