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CN-121991802-A - Adipose-derived stem cell exosome culture device and culture method

CN121991802ACN 121991802 ACN121991802 ACN 121991802ACN-121991802-A

Abstract

The invention belongs to the technical field of adipose-derived stem cell culture, and discloses an adipose-derived stem cell exosome culture device and a culture method, wherein the adipose-derived stem cell exosome culture device comprises an incubator, two independent sealing boxes of a water bath box and a power control box are arranged in the incubator, a first sliding frame is slidably arranged in the water bath box near the upper end, a plurality of mounting plates which are uniformly distributed at intervals are fixedly arranged on the first sliding frame, a constant temperature device is arranged on the inner wall of the water bath box below the mounting plates, a double-output shaft motor is fixedly arranged in the power control box far from the water bath box, a multi-connecting rod device is connected to the double-output shaft ends of the double-output shaft motor in a transmission manner, and the other end of the multi-connecting rod device is fixedly connected with the first sliding frame.

Inventors

  • WANG GUOYING
  • WANG ZHENHUA
  • CAO ZHE
  • XU DONGHUA
  • HUANG JINJIN
  • LONG JIEYI
  • JIANG SHUYUE

Assignees

  • 潍坊市人民医院(潍坊市公共卫生临床中心、潍坊市职业病医院)

Dates

Publication Date
20260508
Application Date
20260210

Claims (10)

  1. 1. A fat stem cell exosome culture device comprises a culture box (1) and is characterized in that a water bath box (110) and a power control box (111) are arranged in the culture box (1), a first sliding frame (21) is slidably arranged at a position close to the upper end in the water bath box (110), a plurality of mounting plates (2) which are uniformly distributed at intervals are fixedly arranged on the first sliding frame (21), a plurality of closed culture dishes (22) which are uniformly distributed at intervals are rotatably arranged on the mounting plates (2), the whole closed culture dishes (22) are completely immersed in constant-temperature bath media, a constant-temperature device (5) is arranged on the inner wall, below the mounting plates (2), of the water bath box (110) and used for accurately stabilizing the constant-temperature bath media at preset culture temperature and feeding back temperature data in real time, a double-output shaft motor (4) is fixedly arranged at a position far away from the water bath box (110), a double-output shaft end of the double-output shaft motor (4) is in a transmission mode, the other ends of the double-output shaft motor (4) are fixedly connected with a plurality of connecting rod devices (3), and the other ends of the connecting rod devices (3) are fixedly connected with the first sliding frame (21) and drive the first sliding frame (21) to reciprocate horizontally.
  2. 2. The fat stem cell exosome culture device according to claim 1, wherein the incubator (1) comprises a case body (11), the case body (11) is divided into a water bath case (110) and a power control case (111) by a middle sealing installation middle partition plate (112), and racks (211) parallel to the length direction of each mounting plate (2) are fixedly installed on the inner wall of the water bath case (110) at positions right below each mounting plate (2).
  3. 3. The fat stem cell exosome culture device according to claim 2, wherein the upper surface of the mounting plate (2) is rotatably provided with a plurality of transmission shafts (213) which are uniformly distributed at intervals, the transmission shafts (213) penetrate through the upper end face and the lower end face of the mounting plate (2), gears (212) are fixedly sleeved at the lower end part of the transmission shafts (213), and all the gears (212) on each mounting plate (2) are in meshed connection with racks (211) below the corresponding mounting plate; the closed culture dish (22) is detachably arranged at the upper end part of the transmission shaft (213).
  4. 4. The culture device of fat stem cell exosome according to claim 3, wherein the constant temperature device (5) comprises a plurality of electric heating rods (51) fixedly arranged on a middle partition board (112) at one side in the water bath box (110), and a plurality of temperature sensors (52) are uniformly arranged in the water bath box (110).
  5. 5. The adipose-derived stem cell exosome culture device according to claim 4, wherein a motor mounting table (32) is vertically arranged at a position of the inner bottom surface of the power control box (111) far away from the middle partition plate (112), and the double-shaft motor (4) is fixedly arranged at the top end of the motor mounting table (32).
  6. 6. The adipose-derived stem cell exosome culture device according to claim 5, wherein the multi-link device (3) comprises a mounting frame (31) fixedly mounted in a power control box (111), lower supporting bearings (34) are fixedly mounted at the bottom end of the mounting frame (31) close to two sides of the double-output shaft motor (4), a multi-link group (33) is connected at the double-output shaft end of the double-output shaft motor (4), and the lower supporting bearings (34) can provide stable rotation fulcrums for swinging of the multi-link group (33).
  7. 7. The culture device of the fat stem cell exosome according to claim 6, wherein the multi-connecting-rod group (33) comprises a crank (331) fixedly installed on a single-side power output shaft of the double-output-shaft motor (4), one end of the crank (331) far away from the motor output shaft is hinged with a rear connecting rod (332), the other end of the rear connecting rod (332) is hinged with one end of a rocker arm (336) to form a movable transmission link, and the lower end of the rocker arm (336) is rotatably installed in an inner ring of a lower supporting bearing (34); the upper ends of the rocker arms (336) at two sides are fixedly connected through an intermediate connecting shaft (35), the upper ends of the rocker arms (336) are further hinged with front connecting rods (333), and one ends of the front connecting rods (333) far away from the rocker arms (336) are hinged with second sliding frames (36).
  8. 8. The adipose-derived stem cell exosome culture device according to claim 7, wherein the two sides of the mounting frame (31) close to the front connecting rod (333) are symmetrically and fixedly provided with U-shaped sliding grooves (334) extending along the horizontal direction, rubber wheels (335) are rotatably arranged at positions of two sides of the second sliding frame (36) corresponding to the U-shaped sliding grooves (334), the rubber wheels (335) and the U-shaped sliding grooves (334) form low-friction rolling fit, and the second sliding frame (36) is ensured to stably slide along the U-shaped sliding grooves (334).
  9. 9. The device for culturing adipose-derived stem cell exosomes according to claim 8, wherein a plurality of connecting plates (23) which are uniformly distributed at intervals are vertically and fixedly arranged at one end of the first sliding frame (21) close to the middle partition plate (112), and one end of the first sliding frame far away from the first sliding frame (21) penetrates through the middle partition plate (112) and is fixedly connected with the second sliding frame (36).
  10. 10. The method for culturing the fat stem cell exosome is based on the device for culturing the fat stem cell exosome according to claim 9, and is characterized by comprising the following steps: s1, cleaning collected adipose tissues, shearing the collected adipose tissues into uniform tissue fragments, fully mixing the uniform tissue fragments with collagenase digestive juice according to a proportion, ensuring that the tissue fragments are in primary contact with digestive enzymes, and aseptically packaging the mixed adipose tissue samples into a plurality of closed culture dishes (22); S2, filling constant-temperature bath medium into the water bath box (110); s3, closing the box cover (12) and locking the box cover through a buckle type locking structure, and setting parameters through a touch panel (6) on the surface of the box body (11); S4, clicking a start button of the touch panel (6), starting the constant temperature device (5) by the control system, controlling the electric heating rod (51) to quickly heat up at maximum power, and simultaneously acquiring temperature data by the plurality of temperature sensors (52) in real time and feeding back the temperature data to the system; s5, when the detected temperature of the constant-temperature bath medium in the water bath tank (110) reaches a threshold value, the control system adjusts the power of the electric heating rod, and the temperature of the constant-temperature bath medium is stabilized at 37 ℃; S6, the control system controls and starts a double-output-shaft motor (4), the double-output-shaft motor (4) converts rotary motion into horizontal reciprocating sliding of a first sliding frame (21) through a multi-connecting rod device (3) according to preset rotating speed, meanwhile, the mounting plate (2) and the closed culture dish (22) are driven to horizontally reciprocate, in the translation process, a gear (212) is meshed and driven along a rack (211), a transmission shaft (213) and the closed culture dish (22) are driven to regularly rotate around a central shaft of the double-output-shaft motor in a positive-negative mode, and fat tissue blocks in the closed culture dish (22) can be fully contacted with digestive enzymes; S7, in the culture process, the control system automatically detects the running state at the same time, and if the abnormality such as overtemperature, leakage and the like occurs, the control system immediately triggers an alarm and executes corresponding safety protection operation; S8, after the cultivation is finished, the control system sends out an ending instruction, the double-output-shaft motor (4) is stopped stably, the electric heating rod (51) stops working, and meanwhile the touch panel (6) displays the completion of the cultivation and sends out a prompt tone; S9, discharging the constant-temperature bath medium, opening a box cover (12), taking out the closed culture dishes (22) one by one, transferring to an ultra-clean workbench, and then carrying out the next treatment; s10, key data in the culture process can be exported through the touch panel (6), so that experiments are convenient to trace, each part in the box body (11) is cleaned and disinfected, and the sterile environment for the next use is ensured.

Description

Adipose-derived stem cell exosome culture device and culture method Technical Field The invention belongs to the technical field of adipose-derived stem cell culture, and particularly relates to an adipose-derived stem cell exosome culture device and a culture method. Background The stem cell exosome is used as a micro vesicle with the diameter of 40-100nm, can carry biomolecules such as lipid, protein, mRNA and the like to realize intercellular communication, and has important application value in the fields of tissue repair, anti-inflammation, anti-fibrosis and the like. The fat stem cells are rich in sources, convenient to obtain and high in secretion of exosomes, become a preferred cell source for exosome preparation, and the efficient culture device and method are core guarantees for improving the extraction rate of the fat stem cells and the yield of the exosomes. In the process of culturing the fat stem cell exosome, the fat tissue block is fully contacted with digestive enzyme through mechanical movement, so that the release of the stem cells from the tissue is promoted, and meanwhile, the culture temperature is precisely controlled to maintain the enzyme activity and the cell stability. The traditional culture method relies on manpower to shake the culture dish, so that the labor intensity is high, the problems of uneven shaking frequency, insufficient contact and the like exist, and the stem cells are low in extraction efficiency and damaged in cell activity. To solve this problem, an automated shaking culture apparatus has appeared in the related art. One of the chinese patents, application number CN202211233827.8, discloses a adipose-derived stem cell exosome culture device. The technical problem that a culture dish cannot be rocked in the process of culturing the fat stem cell exosome by a traditional fat stem cell exosome culture device is solved. Including the incubator, the incubator front end is connected with the door that opens and shuts through the hinge rotation, and the incubator side is provided with power unit, and the incubator internal rotation is connected with the pivot, and the pivot upper end runs through the incubator upper end, and the pivot periphery is provided with rotary mechanism, and rotary mechanism is used for driving the culture dish and rotates, and the pivot is connected with the standing groove through rotary mechanism, has placed the culture dish in the standing groove. The invention can drive the culture dish in the placing groove to shake, thereby achieving the purpose of shaking the culture dish; However, the prior art still has obvious defects that the requirement of efficient large-scale culture is difficult to meet, firstly, the motion mode is single, only revolution shaking (the swinging angle is less than or equal to 30 degrees) of a culture dish around a rotating shaft can be realized, the culture dish cannot generate autorotation, fat tissue blocks are easy to gather in a local area of the culture dish, the contact area with digestive enzyme is small, the problem that the inside of the culture dish is insufficiently digested due to local excessive digestion exists, the stem cell extraction rate is low, secondly, the temperature control mode is simple and crude, the culture dish depends on air heating in an incubator, the temperature fluctuation range is large, enzyme activity is easy to be influenced, the cell release efficiency is further reduced, thirdly, the culture scale is limited, the device is only provided with a single placing groove, the single culture amount is small, and the requirement of industrial mass production cannot be met. Therefore, the existing adipose-derived stem cell exosome culture device has obvious defects in motion structure design, temperature control precision and culture efficiency, and a technical scheme capable of realizing compound motion, precise temperature control and batch culture is needed to solve the problems of uneven contact between adipose tissue blocks and digestive enzymes, low cell extraction rate, insufficient exosome yield and other industry pain points. Disclosure of Invention The invention aims to solve the main technical problems of providing the adipose-derived stem cell exosome culture device and the culture method with simple integral structures, which can improve the temperature control precision in the process of culturing the adipose-derived stem cell exosome, can realize batch culture, and solve the problems of uneven contact between adipose tissue blocks and digestive enzymes and low cell extraction rate. In order to solve the technical problems, the invention provides the following technical scheme: A fat stem cell exosome culture device comprises a culture box, wherein a water bath box and a power control box are arranged in the culture box, a first sliding frame is slidably installed at a position close to the upper end in the water bath box, a plurality of mounting plates which are uniform