CN116426462A - Double dynamic culture method and culture device - Google Patents

Double dynamic culture method and culture device Download PDF

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CN116426462A
CN116426462A CN202310325355.7A CN202310325355A CN116426462A CN 116426462 A CN116426462 A CN 116426462A CN 202310325355 A CN202310325355 A CN 202310325355A CN 116426462 A CN116426462 A CN 116426462A
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hydrogel
elastic membrane
bracket
cell culture
fiber
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李超婧
李皖
王富军
贾东森
任悦颖
毛吉富
王璐
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Donghua University
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Abstract

The invention relates to a double dynamic culture method and a culture device, wherein the double dynamic culture method comprises the following steps: applying downward cyclic compression load to the column-shaped fiber hydrogel bracket after absorbing the cell culture medium I, and utilizing an elastic membrane to assist the fiber hydrogel bracket to rebound in the process, namely realizing double dynamic culture; the fiber hydrogel bracket is of a riser-shaped structure and consists of an inner layer and an outer layer, wherein the inner layer consists of hydrogel and cells dispersed in the hydrogel, and the outer layer consists of a micro-nano fiber bracket, the hydrogel filled in the pores of the micro-nano fiber bracket and the cells dispersed in the hydrogel; the culture device comprises an incubator, a cell culture dish, an elastic membrane, a loading head and a lifting mechanism; the lifting mechanism is used for driving the loading head to do lifting motion. The invention successfully simulates the bearing mode of natural fibrocartilage tissue, and has important significance for regulating and controlling cell behaviors.

Description

Double dynamic culture method and culture device
Technical Field
The invention belongs to the technical field of biomedical engineering, and relates to a double dynamic culture method and a culture device.
Background
The dynamic cell culture simulates the condition that cells in vivo are subjected to dynamic stimulation on the basis of cell culture, applies mechanical stimulation such as stretching, compression, fluid shearing and the like, and further comprises the step of applying dynamic mechanical stimulation in the field of myocardial cell culture to realize the behavior regulation and control of the cells.
Fibrocartilage is a special cartilage tissue which is distributed at intervertebral disc, meniscus, joint disc, pubic symphysis and the like, and has the structural characteristics that collagen fibers are regularly arranged, so that the fibrocartilage tissue is soft, tough and durable. The manner of loading born by fibrocartilage is different from other tissues, when the fibrocartilage is subjected to compressive loading of a body, the compressive loading is converted into the stretching of collagen fibers through a unique collagen fiber structure to carry, and the connected hard bones are protected. Therefore, the dynamic culture of the fibrocartilage cells needs to be performed by taking this special load mode into consideration. Taking one of fibrocartilage, such as meniscus as an example, it can be found that the following problems exist in the existing dual-dynamic culture technique of fibrocartilage cells:
(1) In the prior art, bovine collagen is used for injection molding or a method of combining 3d printing with hydrogel is used for dynamic compression culture, so that the existence of special micro-nano fiber structures of meniscus is ignored, and the special fiber structures can influence the behavior of cells in a natural mechanical environment;
(2) The stretching force of the traditional meniscus scaffold is mostly generated by the expansion of hydrogel holes, and is not consistent with the natural situation;
(3) The prior art cannot flexibly regulate the size of stress stimulus, for example, the compression deformation of a meniscus is about 10% (10% deformation refers to the ratio of the compressed displacement to the height of a bracket per se, for example, the height of hydrogel is 100mm, the compression of 10% is 10mm downwards), and the aperture size of the hydrogel needs to be regulated when the stress stimulus is regulated under the condition of meeting the specified compression deformation, so that the practical operation is very complicated.
Document Physiologically Distributed Loading Patterns Drive the Formation of Zonally Organized Collagen Structures in Tissue-Engineered Meniscus discloses a double dynamic culture device, which applies dynamic compression and stretching to a Niu collagen meniscus bracket to obtain a better dynamic cell culture effect; however, the natural type I collagen fibers in this document are shaped after dissolution, similar to porous hydrogels, and have lost the fibrous structure of the natural meniscus; while meniscus fibers are mainly composed of circumferentially oriented micro-nanofibers, these special fiber structures affect the behavior of cells in natural mechanical environment, and the stretching force of type I collagen is mostly generated by the expansion of hydrogel pores, which is not consistent with the natural situation, so that the type I collagen fibers are stretched in the compression process, and thus the cell stimulation is greatly different from the natural type I collagen fibers.
Document Orchestrated biomechanical, structural, and biochemical stimuli for engineering anisotropic meniscus prepares a toroidal meniscus scaffold by 3D printing in combination with hydrogel, mimics the loading pattern of the natural meniscus by inner layer compression to outer layer stretching (i.e. by expansion of hydrogel pore size), and achieves the effect of differentiation of stem cells in a partition-specific manner; however, the method only adopts a single stretching or compressing means to stimulate the cells, is different from the mechanical stimulation of the fibrocartilage tissue in vivo, and aims at the fibrocartilage tissue engineering, and a dual culture system is required to dynamically culture the cells so as to realize the directional differentiation of the cells and the generation of specific extracellular matrixes.
Therefore, considering that the real fibrocartilage environment has a fiber structure, and the stretching retraction of the fiber can influence the behavior of cells loaded on the fiber, the existing double-culture system needs to be improved, and the stretching of the natural compression load-converted fiber is simulated to perform compression and stretching double-dynamic culture on the cells.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a double dynamic culture method and a culture device.
In order to achieve the above purpose, the invention adopts the following scheme:
a double dynamic culture method applies downward cyclic compression load to a column-shaped fiber hydrogel bracket after absorbing a cell culture medium I, and utilizes an elastic membrane to assist the fiber hydrogel bracket to rebound in the process, namely realizing double dynamic culture;
the fiber hydrogel bracket is of a riser-shaped structure and consists of an inner layer and an outer layer, wherein the inner layer consists of hydrogel and cells dispersed in the hydrogel, and the outer layer consists of a micro-nano fiber bracket, the hydrogel filled in the pores of the micro-nano fiber bracket and the cells dispersed in the hydrogel;
the fiber hydrogel scaffold is used for simulating natural fibrocartilage tissue, wherein the micro-nano fiber scaffold is used for simulating fibers in the natural fibrocartilage tissue; the loading amplitude, the loading frequency and the loading time of the cyclic compression load are set according to the mechanical loading environment of the natural fibrocartilage tissue, for example, if the natural intervertebral disc tissue needs to be simulated, the loading frequency and the loading time are set to be 1HZ, 1h/Day and 5Day/week, and the loading amplitude is 10% deformation; if the loading frequency and time of the natural meniscus tissue are required to be simulated to be 0.5HZ, 4h/Day and 5Day/week, the loading amplitude is 5-10% of deformation, and the mechanical environment of natural walking of a human body is simulated.
Research shows that the arrangement direction of the fibers can influence the cell behavior, and collagen fibers in natural fibrocartilage tissues have certain arrangement direction, so that mechanical stimulation of fiber stretching is required, and the bionic micro-nano fiber scaffold is combined with hydrogel, so that the stimulation of fiber stretching force to cells is realized; in the process of applying cyclic compression load, when the hydrogel is compressed, the hydrogel is stressed to expand to the periphery, free water and substances simultaneously move outwards, fibers in the micro-nano fiber bracket are stressed and stretched, stretching force is applied to cells on the fibers, the purpose of cell partition specific differentiation is achieved, part of load is borne at the same time, and the elastic membrane is stretched to bear the rest load, so that the function of bottom covering is achieved; after the compression of the hydrogel is canceled, the elastic membrane assists the fibrous hydrogel scaffold to rebound.
The micro-nanofiber scaffold is composed of high polymer micro-nanofiber, the structure can be adjusted according to the application, for example, the natural intervertebral disc is formed by stacking I-type collagen fibers in a diamond net shape, the shape of the designed micro-nanofiber scaffold is shown in fig. 2, and the stress condition of the natural intervertebral disc cells can be imitated.
As a preferable technical scheme:
in the double dynamic culture method, the elastic membrane wraps the fiber hydrogel bracket.
In the double dynamic culture method, the cell culture medium II is distributed around the elastic membrane.
According to the double dynamic culture method, the elastic membrane is a microporous elastic membrane, the pore diameter is larger than the substance exchange pore diameter of the cell culture medium I and the cell culture medium II, and is smaller than the maximum diameter of cells; therefore, when the cell culture medium I is compressed, the pore diameter osmotic pressure is overcome, and the cell culture medium II performs real-time substance exchange, so that the nutrition of cells and the waste liquid are ensured, and the cell culture medium I is very beneficial to cell survival even if discharged.
The dual-dynamic culture method has the advantages that the microporous elastic membrane has good stretching performance, the stretching modulus is 1-300 MPa, the elastic recovery performance is excellent, the fatigue resistance performance is good, the mechanical property loss of 10000 times of cyclic stretching under 10% deformation is not more than 5%, the microporous elastic membrane has regular multiple holes, the aperture is adjustable, the aperture is 1-10 microns, and the water osmotic pressure is 10 -13 ～10 -15 m 4 N seconds.
Natural fibrocartilage tissue, such as meniscus, has a large amount of free water, which is compressed by the femur, generates fluid movement, transfers load through pore pressure, and the free water generates maximum stress in the outermost layer, which causes the fibers to stretch; according to the invention, the regulation and control of the osmotic pressure can be realized by replacing microporous elastic membranes with different pore diameters, and the osmotic pressure also determines the size and direction of stress stimulation, so that the mechanical conversion process of compression-fiber stretching is realized, and the stimulation of natural fiber stretching to cells is effectively simulated.
The invention also provides a double dynamic culture device for realizing the double dynamic culture method, which is used for constructing a compression-to-tension load environment of natural fibrocartilage tissue and comprises an incubator, a cell culture dish, an elastic membrane, a loading head and a lifting mechanism;
the cell culture dish is horizontally placed;
the elastic membrane is in a vertical tubular structure and is vertically placed in the cell culture dish, and the elastic membrane and the cell culture dish enclose an outer cavity of the culture medium; the elastic membrane is connected with the cell culture dish and is in a tensioning state;
the loading head is positioned in or above the hollow part of the elastic membrane;
the lifting mechanism is used for driving the loading head to do lifting motion;
the cell culture dish, the elastic membrane and the loading head are all positioned in the incubator, and the elastic membrane and the loading head are biocompatible (i.e. made of a material that is not cytotoxic).
As a preferable technical scheme:
the double dynamic culture device comprises a lifting mechanism, a control mechanism and a control mechanism, wherein the lifting mechanism comprises a servo motor and a transmission shaft which are connected with each other; the servo motor is connected with the incubator, the transmission shaft is vertically arranged, and the loading head is arranged at the lower end of the transmission shaft; the servo motor controls the lifting movement of the loading head through the transmission shaft, the concentration of oxygen and carbon dioxide in the incubator can be flexibly regulated and controlled, meanwhile, the compression frequency and displacement of the loading head can be flexibly regulated and controlled, and the influence of compression displacement parameters on cells can be explored by changing the displacement of the loading head, so that the incubator is flexible and convenient.
The double dynamic culture device has the advantages that the loading head is of a vertical truncated cone-shaped structure, the big end is arranged at the upper part, and the small end is arranged at the lower part, so that the double dynamic culture device can be used for preparing a meniscus-shaped annular fiber hydrogel bracket; alternatively, the loading head is of a vertical cylindrical structure and can be used for preparing the disc-shaped cylindrical fiber hydrogel bracket; or the loading head is of other structures and can be adjusted according to actual requirements.
In the double dynamic culture device, the number of the elastic membranes in the same cell culture dish is 1, and the number of the loading heads is 1.
In the double dynamic culture device, the number of the elastic membranes in the same cell culture dish is n, the number of the loading heads is n, the n loading heads are respectively positioned in or above the hollow parts of the n elastic membranes, the n loading heads are connected with the same lifting mechanism, and n is a positive integer greater than 1; the number of the elastic membrane and the loading head can be adjusted to simultaneously place a plurality of fiber hydrogel brackets, so that the fiber hydrogel brackets are simultaneously cultured, and meanwhile, the cyclic compression load is carried out on the fiber hydrogel brackets, so that the efficiency of the experiment is improved.
The double dynamic culture device is characterized in that the cell culture dish is provided with a liquid exchange port communicated with the outer cavity of the culture medium; the culture medium outer cavity is used for placing the culture medium, and the culture medium can be conveniently replaced by the arrangement of the liquid exchange port, so that the culture medium is prevented from being taken out.
In addition, the invention also provides a double-dynamic culture method adopting the double-dynamic culture device, which comprises the following steps:
(1) After determining the natural fibrocartilage tissue to be prepared, designing the shape and the size of the fiber hydrogel scaffold, and preparing the micro-nano fiber scaffold;
(2) Vertically placing the micro-nano fiber support in the elastic membrane to enable the micro-nano fiber support and the elastic membrane to be in seamless fit;
(3) Adding a cell precursor solution and adjusting the position of a loading head so that the cell precursor solution and the loading head are positioned in the hollow part of the micro-nano fiber bracket, initiating crosslinking to form the fiber hydrogel bracket, wherein the loading head is used for customizing the shape of the hydrogel in the process, and the elastic membrane plays an auxiliary shaping role;
(4) Adding a cell culture medium into the hollow part of the micro-nano fiber bracket and the outer cavity of the culture medium respectively; the hydrogel in the fiber hydrogel bracket is a three-dimensional porous network, is similar to a sponge, can absorb the cell culture medium into the fiber hydrogel bracket, and then exchanges substances with the fresh cell culture medium in the outer cavity of the culture medium; whereas if there is a lack of motivation, mass exchange is extremely slow only in the presence of osmotic pressure;
(5) The loading head is controlled to do lifting movement so as to apply cyclic compression load to the fiber hydrogel bracket, and double dynamic culture is completed after the loading head is finished, wherein the loading amplitude is adjusted by controlling the loading head to descend by different heights; because the hydrogel is an elastic water-containing ball, similar to jelly, the hydrogel can deform when the hydrogel is compressed by the loading head and pressed, and meanwhile, the circumferential expansion is generated under the limitation of the elastic membrane, the circumferential expansion force is transmitted to the micro-nanofiber bracket, and finally, the micro-nanofiber bracket bears the circumferential expansion stress through the tensile force, at the moment, the micro-nanofiber bracket becomes shorter and thicker, and after the loading head is lifted, the micro-nanofiber bracket is rebound and becomes thinner under the help of the elastic membrane, and the hydrogel rebound, so that one mechanical stimulation is completed; the invention promotes substance exchange by bionic mechanical stimulation, and achieves biochemical performance similar to that of natural fibrocartilage tissue.
Advantageous effects
(1) The double dynamic culture method provided by the invention has the advantages that the steps are simple, and the rapid preparation of the bionic shape bracket is realized;
(2) The double dynamic culture method simulates the fiber and cartilage parts of the natural fibrocartilage tissue, combines the double dynamic culture of compression and stretching, applies compression to the cartilage parts, changes the compression to the stretching of the fiber parts, realizes the bearing mode of the natural fibrocartilage tissue, has great significance for regulating and controlling the cell behaviors, and has instructive significance for the subsequent in vivo study;
(3) The double dynamic culture method of the invention not only can adjust the tension force of cells, but also can realize the real-time exchange of inner and outer substances when compressive load is applied, thereby greatly improving the activity of cells;
(4) According to the double dynamic culture device, through the shape design of the loading head, the fiber hydrogel composite bracket with customized shape can be quickly constructed, and the conduction angle of natural compression force can be obtained.
Drawings
FIG. 1 is a schematic diagram of a dual dynamic culture device of the present invention;
FIG. 2 is a schematic diagram of the structure of a cell culture dish according to the invention;
FIGS. 3-5 are schematic views of different types of loaders and culture dishes according to the present invention;
FIG. 6 is a diagram showing the expression of the type I collagen gene in inner and outer cells according to the embodiment of the present invention;
FIG. 7 is a diagram showing the expression of the type II collagen gene in inner and outer cells according to the embodiment of the present invention;
FIG. 8 is an aspect ratio of inner and outer cells in an embodiment of the invention;
FIG. 9 shows the orientation angle of cells in the examples of the present invention (the outer layer is the angle between the cells and the fiber direction, the smaller the orientation angle, the cells grow along the fiber scaffold orientation; the inner layer is the angle between the cells and the horizontal direction, the larger the angle, the more randomly the cells are distributed);
FIG. 10 is a graph of CCK-8 cell viability in an embodiment of the invention;
the device comprises a 1-incubator, a 2-servo motor, a 3-transmission shaft, a 4-loading head, a 4-1-round table-shaped loader, a 4-2-cylindrical loader, a 4-3-multi-head loader, a 5-cell culture dish, a 5-1-liquid exchange port, a 6-elastic membrane, a 7-micro-nanofiber bracket, a hollow part of an 8-micro-nanofiber bracket, a 9-culture medium outer cavity, a 10-annular hydrogel bracket and an 11-cylindrical hydrogel bracket.
Detailed Description
The invention is further described below in conjunction with the detailed description. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
The following examples and comparative examples use the following sources of materials:
(1) Polycaprolactone: from Sigma-Aldrich under the trade designation 900824;
(2) Elastic membrane: is from Jiangsu green Union scientific instruments Co., ltd, and has the trade mark of microporous filter membrane;
(3) Stem cells: derived from the GmbH of the life technology of the Wuhanpranopsis, the commercial brand is rabbit bone marrow mesenchymal stem cells;
(4) Gel precursor solution of gel: the commercial brand is GelMAEFL-GM-30/60/90 from Suzhou Yongqing spring Intelligent equipment Co., ltd;
(5) Cell culture medium i: is available from the growth medium (-micromessentilmium (-MEM) under the trade name Gibco BRL Co.Ltd;
(6) Cell culture medium II: is available from the growth medium (-micromessentilmium (-MEM) under the trade designation Gibco BRL co.
A double dynamic culture method applies downward cyclic compression load to a column-shaped fiber hydrogel bracket after absorbing a cell culture medium I, and utilizes an elastic membrane to assist the fiber hydrogel bracket to rebound in the process, namely realizing double dynamic culture;
the fiber hydrogel bracket is of a riser-shaped structure and comprises an inner layer and an outer layer, wherein the inner layer comprises hydrogel and cells dispersed in the hydrogel, and the outer layer comprises a micro-nanofiber bracket, the hydrogel filled in the pores of the micro-nanofiber bracket and the cells dispersed in the hydrogel;
the fiber hydrogel scaffold is used for simulating natural fibrocartilage tissue, wherein the micro-nano fiber scaffold is used for simulating fibers in the natural fibrocartilage tissue; the loading amplitude, the loading frequency and the loading time of the cyclic compression load are set according to the mechanical loading environment of the natural fibrocartilage tissue;
the elastic membrane wraps the fiber hydrogel bracket, and a cell culture medium II is distributed around the elastic membrane; the preferable elastic membrane is a microporous elastic membrane, the pore diameter is 1-10 microns, the pore diameter is larger than the substance exchange pore diameter of the cell culture medium I and the cell culture medium II and smaller than the maximum diameter of the cells; the tensile modulus (tensile fracture test is carried out by adopting an electric force5500 of America TA company instrument) of the microporous elastic membrane is 1-300 MPa, the mechanical property loss of 10000 times of cyclic stretching under 10% deformation is not more than 5%, and the water osmotic pressure of the microporous elastic membrane is 10 -13 ～10 -15 m 4 N seconds.
A double dynamic culture apparatus for realizing a double dynamic culture method as described above, as shown in fig. 1, comprises an incubator 1, a cell culture dish 5, an elastic membrane 6, a loading head 4 and a lifting mechanism;
the elastic membrane 6 is of a vertical tube-shaped structure, is vertically arranged in the cell culture dish 5 and is connected with the cell culture dish 5 in a tensioning state; as shown in fig. 2, the cell culture dish 5 is horizontally placed, the elastic membrane 6 and the cell culture dish 5 enclose a culture medium outer cavity 9, and the cell culture dish 5 is provided with a liquid exchange port 5-1 communicated with the culture medium outer cavity 9;
as shown in fig. 3, the loading head 4 has a vertical truncated cone-shaped structure, namely, a truncated cone-shaped loader 4-1, the big end of which is at the upper part and the small end of which is at the lower part, and can be used for preparing a meniscus-like annular fiber hydrogel bracket 10; alternatively, as shown in FIG. 5, the loading head 4 is of a vertical cylindrical structure, i.e., a cylindrical loader 4-2, which can be used to prepare an intervertebral disc-shaped cylindrical fibrous hydrogel scaffold 11; alternatively, as shown in fig. 4, the loading head 4 has other structures (such as a multi-head loader 4-3) and can be adjusted according to actual requirements;
in the same cell culture dish 5, the number of the elastic membranes 6 is m, the number of the loading heads 4 is m, the m loading heads 4 are respectively positioned in or above the hollow parts of the m elastic membranes 6, the m loading heads 4 are connected with the same lifting mechanism, and m is a positive integer greater than or equal to 1;
the lifting mechanism comprises a servo motor 2 and a transmission shaft 3 which are connected with each other; the servo motor 2 is connected with the incubator 1, the transmission shaft 3 is vertically arranged, and the loading head 4 is arranged at the lower end of the transmission shaft 3; the servo motor 2 controls the lifting motion of the loading head 4 through the transmission shaft 3;
the cell culture dish 5, the elastic membrane 6 and the loading head 4 are all positioned in the incubator 1, and the elastic membrane 6 and the loading head 4 are biocompatible.
Example 1
A double dynamic culture method adopting the double dynamic culture device is shown in figures 1-3, and comprises the following steps:
(1) After the natural fibrocartilage tissue to be prepared is determined to be an intervertebral disc, designing the shape of a fibrous hydrogel scaffold as shown in figure 2, wherein the dimensions are 10mm in height, 18mm in inner diameter and 20mm in outer diameter, and preparing the polycaprolactone-oriented micro-nano fiber scaffold 7 by adopting an electrostatic spinning method;
(2) Vertically placing the micro-nano fiber support 7 in an elastic membrane 6 with the aperture of 5 mu m, so that the micro-nano fiber support and the elastic membrane are in seamless fit;
(3) Pouring GELMA hydrogel precursor solution loaded with stem cells into the hollow part 8 of the micro-nano fiber support, and adjusting the position of the truncated cone-shaped loader 4-1 to enable the GELMA hydrogel precursor solution to be positioned in the hollow part 8 of the micro-nano fiber support, so as to initiate crosslinking to form the fiber hydrogel support;
(4) Adding a cell culture medium I into the hollow part 8 of the micro-nano fiber bracket, and adding a cell culture medium II into the culture medium outer cavity 9;
(5) The round platform-shaped loader 4-1 is controlled to do lifting motion so as to apply cyclic compression load to the fiber hydrogel bracket, the loading amplitude is set to be 10% for deformation compression, the loading frequencies are set to be 1HZ, 1h/Day and 5Day/week, and after the completion, the double dynamic culture is completed.
After one week, cells attached to the outer layer fibers are in a spindle shape and grow in an oriented manner along the direction of the fibrous scaffold to generate a large amount of type I collagen, which indicates that stem cells are differentiated into fiber-like cells, and the inner layer stem cells highly express type II collagen, which indicates that the stem cells are differentiated into cartilage-like cells and are consistent with natural intervertebral disc tissues.
Comparative example 1
A double dynamic culture method, the steps basically same as example 1, only in that the round table shaped loader is not controlled to do lifting movement, namely, no cyclic compression load is applied to the fiber hydrogel bracket.
Cells attached to outer layer fibers grew along fiber orientation after one week, and the amounts of type I collagen and type II collagen expressed, and the difference in cell phenotype between inner and outer layer cells were not large as in example 1.
Example 2
A double dynamic culture method, the steps basically same as example 1, only in the step (2) of elastic membrane is a nonporous elastic membrane.
After one week, cells attached to the outer layer of fibers were spindle-shaped and grew in an oriented manner along the direction of the fibrous scaffold, producing a large amount of type i collagen, indicating that stem cells exhibited fibroblast-like differentiation, and that the inner layer of stem cells highly expressed type ii collagen, indicating that stem cells exhibited cartilage-like differentiation, consistent with natural disc tissue, but CCK results indicated that cell viability was not as good as example 1.
Comparative example 2
A double dynamic culture method, the steps basically same as example 2, only in that there is no round table shaped loader for lifting movement, i.e. no cyclic compression load is applied to the fiber hydrogel scaffold, and a nonporous elastic membrane is used.
Cells attached to outer layer fibers grew along fiber orientation after one week, and the amounts of type I collagen and type II collagen expressed, and the difference in cell phenotype between inner and outer layer cells were not large as in example 2.
In summary, as shown in fig. 6 to 7, comparing example 1, comparative example 1, example 2 and comparative example 2, it was found that the outer layer type i collagen was greater than the inner layer and the inner layer type ii collagen was greater than the outer layer in all groups of cells, indicating that there was a certain differential differentiation of stem cells due to the bionic fibrous scaffold, but the gene expression amounts and the degree of differential differentiation of comparative example 1 and comparative example 2 were much smaller than those of example 1 and example 2 due to the bionic dynamic stimulation.
As shown in fig. 8, the aspect ratio of the outer cells of all groups was greater than that of the inner layer, indicating that the outer cells were spindle-shaped and the inner cells were round, further demonstrating that the fibrous scaffold induced differentiation of cells toward the fibers. In addition, the difference between the inner layer and the outer layer is further increased by dynamic culture, as shown in example 1, the aspect ratio of the outer layer cells is far greater than that of the inner layer cells, the aspect ratio of the outer layer cells is maximum, and the aspect ratio of the inner layer cells is minimum, which means that the difference between the inner layer and the outer layer is more obvious in the example, the outer layer is differentiated towards the fiber, and the inner layer is differentiated towards the cartilage, which is slightly higher than that in the example 2, because the elastic membrane is adopted to improve the activity of the cells, and the differentiation degree of the cells is further improved.
As shown in fig. 9, all groups of outer cells had very small orientation angles with the fibrous scaffold, indicating that the outer cells were aligned along the fibers, and that the fiber orientation induced cell orientation, which was consistent with the orientation direction of the outer cells of the natural disc. The orientation angle of the inner layer cells is larger, which indicates that the inner layer cells are arranged randomly and accords with the random arrangement mode of the chondrocytes at the natural intervertebral disc.
As shown in fig. 10, the level of cell viability of example 1 is highest, the level of example 2 is lower than that of example 1, and the level of comparative example 1 is similar to and lowest than that of comparative example 2, because example 1 adopts a porous elastic membrane to ensure the real-time exchange of nutrients, and adopts a loading head to do cyclic compression load applied by lifting motion, so that the superposition effect is generated between the real-time exchange of nutrients and mechanical stimulation, the exchange efficiency of nutrients is greatly promoted, and the cell viability and the increment level are highest.

Claims (10)

1. A double dynamic culture method is characterized in that a downward cyclic compression load is applied to a column-shaped fiber hydrogel bracket after absorbing a cell culture medium I, and an elastic membrane is utilized to assist the fiber hydrogel bracket to rebound in the process, so that double dynamic culture is realized;
the fiber hydrogel bracket is of a riser-shaped structure and consists of an inner layer and an outer layer, wherein the inner layer consists of hydrogel and cells dispersed in the hydrogel, and the outer layer consists of a micro-nano fiber bracket, the hydrogel filled in the pores of the micro-nano fiber bracket and the cells dispersed in the hydrogel;
the fiber hydrogel scaffold is used for simulating natural fibrocartilage tissue, wherein the micro-nano fiber scaffold is used for simulating fibers in the natural fibrocartilage tissue; the loading amplitude, loading frequency and loading time of the cyclic compression load are set according to the mechanical loading environment of the natural fibrocartilage tissue.
2. The method of claim 1, wherein the elastic membrane encapsulates the fibrous hydrogel scaffold.
3. A dual dynamic culture method according to claim 2, wherein the elastic membrane is surrounded by cell culture medium II.
4. A dual dynamic culture method according to claim 3, wherein the elastic membrane is a microporous elastic membrane having a pore size larger than the mass exchange pore size of the cell culture medium I and the cell culture medium II and smaller than the maximum diameter of the cells.
5. The method according to claim 4, wherein the microporous elastic membrane has a tensile modulus of 1 to 300MPa, a mechanical property loss of 10000 times under 10% deformation of not more than 5%, a pore diameter of 1 to 10 μm, and a water osmotic pressure of 10 -13 ～10 -15 m 4 N seconds.
6. A double dynamic culture device for realizing a double dynamic culture method according to any one of claims 1 to 5, comprising an incubator (1), a cell culture dish (5), an elastic membrane (6), a loading head (4) and a lifting mechanism;
the cell culture dish (5) is horizontally placed;
the elastic membrane (6) is of a vertical tube-shaped structure and is vertically placed in the cell culture dish (5), and the elastic membrane and the cell culture dish enclose a culture medium outer cavity (9); the elastic membrane (6) is connected with the cell culture dish (5) and is in a tensioning state;
the loading head (4) is positioned in or above the hollow part of the elastic membrane (6);
the lifting mechanism is used for driving the loading head (4) to do lifting movement;
the cell culture dish (5), the elastic membrane (6) and the loading head (4) are all positioned in the incubator (1), and the elastic membrane (6) and the loading head (4) have biocompatibility.
7. A double dynamic culture device according to claim 6, wherein the lifting mechanism comprises a servo motor (2) and a transmission shaft (3) which are connected with each other; the servo motor (2) is connected with the incubator (1), the transmission shaft (3) is vertically arranged, and the loading head (4) is arranged at the lower end of the transmission shaft (3).
8. A dual dynamic culture device according to claim 6, wherein the number of elastic membranes (6) in the same cell culture dish (5) is 1 and the number of loading heads (4) is 1; or in the same cell culture dish (5), the number of the elastic membranes (6) is n, the number of the loading heads (4) is n, the n loading heads (4) are respectively positioned in or above the hollow parts of the n elastic membranes (6), the n loading heads (4) are connected with the same lifting mechanism, and n is a positive integer greater than 1.
9. A dual dynamic culture device according to claim 6, wherein the cell culture dish (5) is provided with a liquid exchange port (5-1) which is communicated with the culture medium outer cavity (9).
10. A double dynamic culture method using a double dynamic culture device according to any one of claims 6 to 9, characterized by the steps of:
(1) After the natural fibrocartilage tissue to be prepared is determined, designing the shape and the size of a fibrous hydrogel scaffold, and preparing a micro-nano fiber scaffold (7);
(2) Vertically placing the micro-nano fiber support (7) in the elastic membrane (6) to enable the micro-nano fiber support and the elastic membrane to be in seamless fit;
(3) Adding a cell precursor solution and adjusting the position of the loading head (4) so that the cell precursor solution and the loading head are positioned in the hollow part (8) of the micro-nano fiber bracket (7), and initiating crosslinking to form a fiber hydrogel bracket;
(4) Adding a cell culture medium into the hollow part (8) of the micro-nano fiber bracket (7) and the culture medium outer cavity (9) respectively;
(5) The loading head (4) is controlled to do lifting movement so as to apply cyclic compression load to the fiber hydrogel bracket, and after the completion, the double dynamic culture is completed.
CN202310325355.7A 2023-03-30 2023-03-30 Double dynamic culture method and culture device Pending CN116426462A (en)

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* Cited by examiner, † Cited by third party
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CN116769565A (en) * 2023-08-28 2023-09-19 北京大学口腔医学院 For regulating intracellular m 6 Method for A methylation modification
CN116836781A (en) * 2023-08-28 2023-10-03 北京大学口腔医学院 Force application device, female die and die for simulating mechanical action of cell methylation modification in tooth germ development

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116769565A (en) * 2023-08-28 2023-09-19 北京大学口腔医学院 For regulating intracellular m 6 Method for A methylation modification
CN116836781A (en) * 2023-08-28 2023-10-03 北京大学口腔医学院 Force application device, female die and die for simulating mechanical action of cell methylation modification in tooth germ development
CN116769565B (en) * 2023-08-28 2023-11-14 北京大学口腔医学院 For regulating intracellular m 6 Method for A methylation modification
CN116836781B (en) * 2023-08-28 2023-12-19 北京大学口腔医学院 Force application device, female die and die for simulating mechanical action of cell methylation modification in tooth germ development

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