A cell circulation seeding device and method for tissue engineering scaffolds

Abstract

The application belongs to the field of biomedical experiment instruments, and discloses a cell circulation inoculation device and method for tissue engineering scaffolds. The cell circulation inoculation device comprises a fixing device, an inoculation platform and a circulation device. The inoculation platform comprises an inoculation cavity, a scaffold fixing clamp, a scaffold fixing clamp groove, a sampling door, an inoculation hole, a rotating shaft, a belt and a motor. The circulation device comprises a piston device, a syringe pump, a three-way valve, a one-way valve and a catheter. The tissue engineering scaffold is fixed on the inoculation platform, the motor is turned on to drive the tissue engineering scaffold to rotate, cell suspension is added into the cell liquid cavity in the circulation device, the syringe pump drives the piston to make the cell suspension pass through the tissue engineering scaffold, the cells are adsorbed on the surface of the scaffold, the dropped cell suspension is returned to the cell liquid cavity through the piston, a dynamic circulation loop is formed, and the tissue engineering scaffold is taken out from the sampling door after inoculation. The application can significantly improve the cell inoculation efficiency and the uniformity of cell distribution on the scaffold material, and has simple structure and convenient operation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical experimental instruments, and specifically relates to a cell circulation seeding device and method for tissue engineering scaffolds. Background Technology

[0002] Tissue engineering strategies focus on restoring pathologically altered tissue structures by combining transplanted cells with supporting scaffolds, offering the potential for regeneration of virtually all tissues and organs in the human body. A major challenge in scaffold-based approaches within tissue engineering is the efficient and uniform distribution of high-density cells throughout the scaffold volume. High-density cell implantation into scaffolds is associated with enhanced tissue formation, including higher extracellular matrix production rates, stronger tissue structures, and shorter tissue formation times. Furthermore, the initial distribution of cells within the scaffold after seeding is correlated with the distribution of tissue subsequently formed within the scaffold construct, suggesting that uniform cell seeding lays the foundation for uniform tissue generation.

[0003] Current cell seeding methods include static and dynamic methods. Static loading of cells onto scaffolds is the most common seeding method, but this method has low seeding efficiency, with only about 25% of cells adhering to the scaffold. It also tends to cause uneven cell distribution on the scaffold, making it prone to contamination and difficult to transform into large-scale production. This is because the process relies on manual operation, and the seeding effect is highly dependent on the operator's skill. Dynamic seeding includes stirring, magnetic force, rotation, centrifugation, etc. Although these seeding methods require fewer cells and ensure even distribution on the scaffold, they still have problems such as low seeding efficiency and difficulty in removing the scaffold for some small scaffolds with large gaps.

[0004] Currently, the most common cell dynamic seeding and culture systems are stirred cell, rotating wall, and perfusion bioreactors. Among these, the above devices generally suffer from low seed cell utilization, uneven cell distribution, inability to meet the seeding requirements of tissue engineering scaffolds of different shapes and sizes, and difficulty in scaffold removal; in addition, some of the above devices have very complex structures, are difficult to manufacture, and have high costs. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a cell circulation seeding device for tissue engineering scaffolds. This device has a simple structure, high seeding efficiency, high cell utilization rate, and can be reused repeatedly, achieving high utilization rate of seed cells and uniform seeding of cells on tissue engineering scaffolds.

[0006] Another objective of this invention is to provide a cell circulation seeding method for tissue engineering scaffolds; this method achieves quantitative and uniform seeding of cells, improves cell utilization, and uses a simple and easy-to-operate device.

[0007] The objective of this invention is achieved through the following solution:

[0008] A cell circulation seeding device for tissue engineering scaffolds includes a fixation device, a seeding platform, and a circulation device connected in sequence.

[0009] The fixing device includes a base plate, a support rod, and a fixing clamp. The fixing clamp can be moved up and down on the support rod to adjust its position. The fixing clamp and the fixing part on the side of the inoculation platform are detachably connected.

[0010] The inoculation platform includes an inoculation chamber, a platform fixing part, a support fixing clamp, a support fixing clamp groove, an inoculation hole, a rotating shaft, a belt, a motor, and a sampling door. The platform fixing part is located on the left side of the inoculation platform and is detachably connected to the fixing clamp of the fixing device. The support fixing clamp is detachably connected to the support fixing clamp groove. The support fixing clamp is used to fix the tissue engineering scaffold and is detachable. After the tissue engineering scaffold is fixed by the support fixing clamp, it is suspended in the inoculation chamber. The right side of the inoculation platform has a rotating shaft, which is detachably connected to the support fixing clamp groove. The motor drives the rotating shaft to rotate via a belt, thereby driving the tissue engineering scaffold to rotate as well. The inoculation platform has an inoculation hole at the top, which is sealed with a connector. The other end of the connector is connected to a conduit. The inoculation platform has a through hole at the bottom, which is sealed with a connector. The other end of the connector is connected to a conduit. The front of the inoculation platform has a sampling door with a round handle.

[0011] The circulation device includes a conduit, a three-way valve, a first one-way valve, a second one-way valve, a piston assembly, a dispensing chamber, and an injection pump. Each end of the three-way valve is connected to a one-way valve, and one end is connected to the piston assembly via a Luer connector. The first one-way valve is connected to the dispensing chamber via a conduit, and the dispensing chamber is connected to the inoculation port of the inoculation platform via a conduit. The second one-way valve is connected to the through-hole of the inoculation platform via a conduit. The piston assembly consists of a piston, a piston cylinder, and a piston handle. The space formed by the piston and the inner wall of the piston cylinder is a cell fluid chamber, with an inlet above the cell fluid chamber. The piston handle is connected to the fixing clamp of the injection pump, and the injection pump can drive the piston to reciprocate.

[0012] Preferably, the support fixing clip in the inoculation platform has a snap-fit ​​structure that allows for detachable connection between the support fixing clip groove and the support fixing clip.

[0013] Preferably, the inoculation platform has multiple bracket fixing slots, with one bracket fixing slot corresponding to one bracket fixing clip.

[0014] Preferably, the support clamp in the inoculation platform can be removed from the sampling door of the inoculation platform;

[0015] Preferably, the conduit in the circulation device is a breathable flexible tube with a diameter of 2-4 mm;

[0016] Preferably, the inoculation platform and circulation device can be sterilized and reused;

[0017] Preferably, the inoculation chamber is a cubic transparent container;

[0018] Preferably, the motor in the inoculation platform can be set to rotate at a frequency ranging from 0.5 r / min to 2 r / min;

[0019] Preferably, the injection pump in the circulation device can be adjusted to set the circulation inoculation time, with the number of cycles ranging from 20 to 40, and the inoculation time for each cycle being the infusion and extraction time, ranging from 3 to 6 minutes.

[0020] A method for circulating cell seeding for tissue engineering scaffolds, comprising the following steps:

[0021] Cell suspension is added to the cell fluid chamber of the aforementioned cell circulation inoculation device; the scaffold clamp fixes the tissue engineering scaffold in the inoculation chamber, the motor is turned on to make the tissue engineering scaffold rotate slowly, the injection pump parameters are set, the injection pump is started, the cell suspension drips, some cells adhere to the scaffold surface, and the fallen cells return to the cell fluid chamber. After several cycles of inoculation, the sampling door of the inoculation platform is opened, the tissue engineering scaffold is removed, and the inoculation is completed.

[0022] The features of this invention are:

[0023] (1) Cells that have fallen off tissue engineering scaffolds can be reused and circulated for seeding, thereby improving cell utilization.

[0024] (2) It can precisely adjust the number and density of cells, the number of cycles and the rate of the injection pump, and efficiently seed cells in a short time.

[0025] (3) The present invention can rotate the tissue engineering scaffold so that cells are evenly seeded on the three-dimensional tissue engineering scaffold.

[0026] (4) The present invention has three inoculation holes, which can inoculate three supports at the same time, thereby improving experimental efficiency.

[0027] (5) The device of the present invention has a simple structure and small size, and can be directly placed in a cell culture box for cell inoculation.

[0028] Working principle of the invention:

[0029] The tissue engineering scaffold is inserted into the scaffold clamp and fixed in the scaffold clamp slot, connected to the rotating shaft. The motor is turned on, and the rotating shaft is driven to rotate via a belt, thereby rotating the scaffold. Cell suspension is added to the cell fluid chamber, and the inoculation pump inoculation program is started: the inoculation pump push rod drives the piston to move, pumping the cell suspension into the reservoir through the one-way valve. The cell suspension is sprayed onto the tissue engineering scaffold through three inoculation orifices. Some of the cell suspension remains on the scaffold, and some drips off. At this time, the inoculation pump push rod drives the piston to move in the opposite direction, applying negative pressure, and drawing the dripping cell suspension back into the inoculation chamber through the one-way valve, forming a circulating inoculation loop.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] (1) This device can inoculate cells in a closed sterile environment to avoid contamination. The device is small in size and can be placed in an incubator for inoculation, reducing cell death rate.

[0032] (2) This device can precisely control cell seeding density and seeding rate, and can simultaneously place multiple scaffolds to standardize and streamline the seeding process, reduce experimental errors, and make it efficient and convenient to use.

[0033] (3) This device can collect cells that fall off during the inoculation process, realize dynamic cyclic inoculation, improve cell utilization, save manpower, and improve inoculation efficiency.

[0034] (4) This device can be sterilized and reused.

[0035] (5) This device has multiple support clamps of different specifications, and can be used for different tissue engineering supports.

[0036] (6) Avoid the problem of unscientific research models caused by uneven mass transfer between cells in the center of the three-dimensional scaffold and cells on the sides of the scaffold due to the irregularity of the scaffold in traditional inoculation systems. Attached Figure Description

[0037] Figure 1 This is an overall diagram of a cell circulation seeding device system for tissue engineering scaffolds;

[0038] Figure 2 A 3D view of the vaccination platform;

[0039] Figure 3 A top view of the vaccination platform;

[0040] Figure 4 A cross-sectional view of the inoculation platform;

[0041] Among them, 1-base plate, 2-support rod, 3-fixing clamp, 4-injection pump, 5-piston handle, 6-piston, 7-cell fluid chamber, 8-piston cylinder, 9-inlet, 10-three-way valve, 11-first one-way valve, 12-dispensing chamber, 13-inoculation hole, 14-support fixing clamp, 15-support fixing clamp groove, 16-rotating shaft, 17-belt, 18-motor, 19-fixing part, 20-sampling gate, 21-conduit, 22-second one-way valve, 23-inoculation chamber. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0043] like Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown, the present invention provides a cell circulation seeding device for tissue engineering scaffolds, specifically including a fixation device, a seeding platform, and a circulation device.

[0044] The fixing device includes a base plate 1, a support rod 2, and a fixing clamp 3. The fixing clamp can be moved up and down to adjust its position. The fixing clamp 3 and the fixing part 19 on the side of the inoculation platform are detachably fixed.

[0045] The inoculation platform includes a fixing part 19 on the side of the inoculation platform, an inoculation chamber 23, a stent fixing clip 14, a stent fixing clip groove 15, an inoculation hole 13, a rotating shaft 16, a belt 17, a motor 18, and a sampling gate 20. The stent fixing clip 14 is used to fix the tissue engineering scaffold and is detachable. The stent fixing clip 14 is available in different specifications to accommodate tissue engineering scaffolds of different shapes and sizes. The motor 18 drives the rotating shaft 16 to rotate via the belt 17, thereby driving the stent fixing clip groove 15 to rotate. The stent fixing clip groove 15 fixes the stent fixing clip 14 through a snap-fit ​​structure, thus causing the tissue engineering scaffold to rotate in mid-air. The stent fixing clip 14 is inserted into or removed from the inoculation platform through the sampling gate 20. The inoculation chamber 23 is preferably a cubic transparent container to facilitate observation of the internal state of the tissue engineering scaffold.

[0046] The inoculation platform has three inoculation holes 13 at the top, which are sealed with Luer connectors. The other end of the Luer connector is connected to a conduit 21 for the cell suspension to enter the inoculation platform. There is a through hole at the bottom of the inoculation platform, which is sealed with a Luer connector. The other end of the Luer connector is connected to a conduit 21, through which cells that have not adhered to the tissue engineering scaffold return to the cell fluid chamber 7.

[0047] The circulation device includes a conduit 21, a three-way valve 10, two one-way valves 11 and 22, a piston assembly, an inlet 9, a dispensing chamber 12, and an injection pump 4. The piston assembly consists of a piston 6, a piston cylinder 8, and a piston handle 5. Each end of the three-way valve 10 is connected to a one-way valve, and the other end is connected to the piston assembly via a Luer connector. The one-way valve 11 is connected to the dispensing chamber 12 via a conduit, and the dispensing chamber 12 is then connected to the inoculation port 13 of the inoculation platform via a conduit. The one-way valve 22 is connected to the through-hole of the inoculation platform via a conduit 21, which is a breathable conduit with a diameter of 2-4 mm. The space formed by the piston and the inner wall of the piston cylinder is a cell fluid chamber 7. An inlet 9 is located above the cell fluid chamber, through which cell suspension is added. The piston handle 5 is fixed in a slot in the injection pump 4, which drives the piston 6 to reciprocate, allowing the cell suspension to reciprocate through the tissue engineering scaffold. The speed and number of cycles of inoculation can be set in the injection pump 4.

[0048] This invention can also be expanded to include multiple inoculation ports 13 and multiple scaffold clamps 14 to achieve high-throughput inoculation of tissue engineering scaffolds. Specifically, the number of inoculation ports is set to multiple, and the number of scaffold clamps is the same as the number of inoculation ports, with corresponding positions. The entire device is sterilized before use and can be reused after cleaning and sterilization.

[0049] Example 1

[0050] A method for circulating cell seeding for tissue engineering scaffolds includes the following steps:

[0051] Step 1: Clean and sterilize the device.

[0052] Step 2: Wet the hollow fiber scaffold with culture medium, and then install the three hollow fiber scaffolds onto the scaffold fixing clip 14 respectively. Fix the scaffold fixing clip 14 to the scaffold fixing clip groove 15 through the sampling gate 20.

[0053] Step 3: The prepared human renal cortical proximal tubule epithelial cell suspension is added to the cell fluid chamber 7 through inlet 9. The entire apparatus, except for the syringe pump 4, is placed in a cell culture incubator. The motor is started, and the parameters are set to 1 revolution per minute to slowly rotate the hollow fiber scaffold. The syringe pump is started, and the parameters are set to 2 ml of cell suspension, 4 minutes of perfusion, 1 minute of aspiration, and 30 cycles. The syringe pump pushes the piston forward, and the cell suspension enters the inoculation well 13 through the one-way valve 11. After passing through the hollow fiber scaffold, some cells adhere to the scaffold, while some cell suspension falls through the gaps in the scaffold. The syringe pump pulls the piston backward, and the cell suspension returns to the cell fluid chamber 7. This cycle is repeated 30 times as described above.

[0054] Step 4: After the inoculation cycle is completed, remove the stent fixation clip 14 and then remove the three hollow fiber stents from the stent fixation clip 14.

[0055] Step 5: Place the hollow fiber scaffold inoculated with cells into the culture medium for further culture.

[0056] Example 2

[0057] A method for circulating cell seeding for tissue engineering scaffolds includes the following steps:

[0058] Step 1: Clean and sterilize the device.

[0059] Step 2: Wet the osteogenic hydrogel with culture medium, and then install the three osteogenic hydrogels onto the support clamp 14 respectively. Fix the support clamp 14 to the support clamp groove 15 through the sampling gate 20.

[0060] Step 3: Add the prepared bone marrow mesenchymal stem cell suspension to the cell fluid chamber 7 through inlet 9. Place the entire device, except for the injection pump 4, into a cell culture incubator. Start the motor and set the parameters to 1 revolution per minute to slowly rotate the osteogenic hydrogel scaffold. Start the injection pump and set the parameters to 2 ml of cell suspension, 4 minutes of perfusion, 1 minute of aspiration, and 30 cycles. The injection pump pushes the piston forward, and the cell suspension enters the inoculation well 13 through the one-way valve 11. After passing through the osteogenic hydrogel scaffold, some cells adhere to the osteogenic hydrogel scaffold, while some cell suspension falls through the gaps in the scaffold. The injection pump pulls the piston backward, and the cell suspension returns to the cell fluid chamber 7. Repeat the cycle 30 times as described above.

[0061] Step 4: After the inoculation cycle is completed, remove the scaffold fixation clip 14 and then remove the three osteogenic hydrogels from the scaffold fixation clip 14.

[0062] Step 5: Place the cell-inoculated osteogenic hydrogel scaffold into the culture medium for further culture.

[0063] Example 3

[0064] A method for circulating cell seeding for tissue engineering scaffolds includes the following steps:

[0065] Step 1: Clean and sterilize the device.

[0066] Step 2: Wet the skin tissue engineering scaffold with culture medium, and then install the three skin tissue engineering scaffolds onto the scaffold fixing clip 14 respectively. Fix the scaffold fixing clip 14 to the scaffold fixing clip groove 15 through the sampling gate 20.

[0067] Step 3: Add the prepared fibroblast suspension to the cell fluid chamber 7 through inlet 9. Place the entire device, except for the injection pump 4, into the cell culture incubator. Start the motor and set the parameters to 1 revolution per minute to slowly rotate the skin tissue engineering scaffold. Start the injection pump and set the parameters to 2 ml of cell suspension, 4 minutes of perfusion, 1 minute of aspiration, and 30 cycles. The injection pump pushes the piston forward, and the cell suspension enters the inoculation well 13 through the one-way valve 11. After passing through the skin tissue engineering scaffold, some cells adhere to the scaffold, while some cell suspension falls through the gaps in the scaffold. The injection pump pulls the piston backward, and the cell suspension returns to the cell fluid chamber 7. Repeat the cycle 30 times as described above.

[0068] Step 4: After the inoculation cycle is completed, remove the scaffold fixation clip 14 and then remove the three skin tissue engineering scaffolds from the scaffold fixation clip 14.

[0069] Step 5: Place the cell-inoculated skin tissue engineering scaffold into the culture medium for further culture.

[0070] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A cell circulation seeding device for tissue engineering scaffolds, characterized in that, Includes fixation device, inoculation platform, and circulation device; The fixing device includes a base plate, a support rod, and a fixing clamp. The fixing clamp can be moved up and down on the support rod to adjust its position. The fixing clamp and the fixing part on the side of the inoculation platform can be detachably fixed. The inoculation platform includes an inoculation chamber, a platform fixing part, a support fixing clamp, a support fixing clamp groove, an inoculation hole, a rotating shaft, a belt, a motor, and a sampling door. The support fixing clamp is detachably connected to the support fixing clamp groove. The support fixing clamp is used to fix the tissue engineering scaffold and is detachable. After being fixed by the support fixing clamp, the tissue engineering scaffold is suspended in the inoculation chamber. A rotating shaft is located on the right side of the inoculation platform, and the rotating shaft is detachably connected to the support fixing clamp groove. The motor drives the rotating shaft to rotate via a belt, thereby causing the tissue engineering scaffold to rotate as well. An inoculation hole is located above the inoculation platform, sealed with a connector, the other end of which is connected to a conduit. A through hole is located below the inoculation platform, sealed with a connector, the other end of which is connected to a conduit. A sampling door is located on the front of the inoculation platform. The circulation device includes a conduit, a three-way valve, a one-way valve, a piston assembly, a dispensing chamber, and an injection pump. Each end of the three-way valve is connected to a one-way valve, and one end is connected to the piston assembly via a connector. The first one-way valve is connected to the dispensing chamber via a conduit, and the dispensing chamber is connected to the inoculation port of the inoculation platform via a conduit. The second one-way valve is connected to the through-hole of the inoculation platform via a conduit. The piston assembly consists of a piston, a piston cylinder, and a piston handle. The space formed by the piston and the inner wall of the piston cylinder is a cell fluid chamber, with an inlet above the cell fluid chamber. The piston handle is connected to the fixing clamp of the injection pump, and the injection pump can drive the piston to reciprocate. The injection pump in the circulation device can adjust the circulation time, with the number of cycles ranging from 20 to 40. The inoculation time for each cycle is the infusion and extraction time, ranging from 3 to 6 minutes. The motor in the inoculation platform is set to rotate at a frequency ranging from 0.5 r / min to 2 r / min.

2. The cell circulation seeding device for tissue engineering scaffolds according to claim 1, characterized in that: In the inoculation platform, the platform fixing part is located on the left side of the inoculation platform and is detachably connected to the fixing clamp of the fixing device.

3. The cell circulation seeding device for tissue engineering scaffolds according to claim 1, characterized in that: The second one-way valve is connected to the inoculation platform through a conduit.

4. The cell circulation seeding device for tissue engineering scaffolds according to claim 1, characterized in that: The support fixing clip in the inoculation platform is provided with a buckle structure for detachable connection with the support fixing clip groove.

5. The cell circulation seeding device for tissue engineering scaffolds according to claim 1, characterized in that: The inoculation platform has multiple bracket fixing slots, with one bracket fixing slot corresponding to one bracket fixing clamp.

6. The cell circulation seeding device for tissue engineering scaffolds according to claim 1, characterized in that: The support clamp in the inoculation platform is removed from the sampling door of the inoculation platform; The inoculation platform and circulation device can be reused after sterilization. The inoculation chamber is a cubic transparent container.

7. The cell circulation seeding device for tissue engineering scaffolds according to claim 1, characterized in that: The conduit in the circulation device is a breathable flexible tube with a diameter of 2-4 mm.

8. A method for circulating cell seeding for tissue engineering scaffolds, characterized in that, Includes the following steps: A cell suspension is added to the cell fluid chamber of the cell circulating inoculation device according to any one of claims 1 to 7; The scaffold clamp secures the tissue engineering scaffold to the inoculation platform. The motor is turned on, causing the tissue engineering scaffold to rotate slowly. The injection pump parameters are set, and the injection pump is started. The cell suspension drips, and some cells adhere to the scaffold surface. The fallen cells return to the cell fluid chamber. After the inoculation is completed, the sampling door of the inoculation platform is opened, and the tissue engineering scaffold is removed, completing the inoculation process.

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