CN111925984A

CN111925984A - Cell co-culture system and construction method and application thereof

Info

Publication number: CN111925984A
Application number: CN202010852067.3A
Authority: CN
Inventors: 周德志
Original assignee: East China Institute Of Digital Medical Engineering
Current assignee: East China Institute Of Digital Medical Engineering
Priority date: 2020-08-21
Filing date: 2020-08-21
Publication date: 2020-11-13

Abstract

The invention relates to a cell co-culture system and a construction method and application thereof, belonging to the field of biological three-dimensional printing. The cell co-culture system comprises: a hydrogel scaffold containing first cultured cells therein; a liquid matrix in which the hydrogel scaffold is disposed such that the first cultured cells are present in the liquid matrix, the liquid matrix containing the second cultured cells therein. The construction method of the cell co-culture system comprises the following steps: the method comprises a raw material preparation step, a printing step, a crosslinking step and a mixing step. The hydrogel scaffold can simulate the in-vivo three-dimensional environment required by cell co-culture, has stable structure and can support long-time co-culture. Compared with a microsphere co-culture system, the cell co-culture system has better structural continuity and integrity; the hydrogel scaffold can be constructed into different shapes and wrapped by different types of first culture cells according to requirements, so that a customized co-culture model can be constructed conveniently.

Description

Cell co-culture system and construction method and application thereof

Technical Field

The invention belongs to the field of biological three-dimensional printing, and particularly relates to a cell co-culture system and a construction method and application thereof.

Background

In animal bodies, most tissues are composed of two or more cells, and in order to establish a culture system more similar to the in vivo environment, so that the cells can communicate information with each other and support each other for growth and proliferation, a cell co-culture technology is developed on the basis of the cell culture technology. The cell co-culture technology is to mix two or more kinds of cells (from the same tissue or different tissues) to establish an in vitro cell co-culture system, so as to simulate the in vivo environment to the maximum extent, maintain the in vivo properties, and realize the purposes of in vitro cell induced differentiation, in vitro cell amplification, cell-to-cell interaction research and the like.

Feeder layer cells are single-layer cells which are obtained after certain chemical or radiation treatment and have inhibited mitotic process, and the cells can not divide and proliferate and still maintain metabolic activity; the feeder layer cells and the target cells are cultured together, and compared with the single cell in vitro culture, the feeder layer cells are added, so that the target cells can better maintain the original form and function expression of the cells in vitro for a long time, and the feeder layer cells have better effects on the performance maintenance and proliferation of the target cells.

Currently, cell co-culture systems can be classified into the following two types according to the contact mode of cells: contact culture and indirect culture. The contact culture is to co-culture two kinds of cells in the same culture dish in certain ratio under proper conditions. For example, a three-dimensional scaffold is prepared in advance using a material having good biocompatibility, and feeder cells and cells of interest are seeded on the three-dimensional scaffold to establish a cell co-culture system. Although the method can simulate the three-dimensional growth environment of cells in vitro, the two cells are in direct contact, the difficulty in collecting the target cells is increased by the mixing of feeder layer cells and the target cells, and the target cells can be subjected to downstream research or application after being purified.

Indirect culture, i.e.the two cells of a co-culture system are not in direct contact, the effect of one cell on the other is achieved by the interaction of paracrine cytokines. In the early indirect culture, feeder cells were cultured in advance, and the supernatant after the culture was collected as a medium component of the objective cells. The indirect culture method can avoid introducing feeder cells in the culture process of target cells, but the method is complex in operation and needs repeated experimental operations on two kinds of cells, such as extraction, filtration, replacement and the like of a culture medium. Furthermore, this approach does not mimic the environment of the cells in vivo, and there is no communication between feeder cells and the cells of interest. The culture dish is divided into an upper chamber and a lower chamber by using a Transwell chamber, and two kinds of cells are respectively planted in the upper chamber and the lower chamber; since the Transwell chamber has a permeable membrane with 0.1-12 μm micropores, the cells planted in the upper and lower chambers can exchange substances through the micropores. The method can reduce experimental operation steps in the process of cell co-culture, and simultaneously, the two cells can be mutually regulated in real time. However, in the co-culture process, the material exchange in the upper and lower layers of the medium is limited due to the limitation of the area of action of the membrane of the Transwell chamber, and there is a phenomenon of a material concentration gradient. Therefore, in the indirect culture, neither the supernatant culture method in which feeder cells are cultured alone nor the layer culture method using a Transwell chamber can simulate the microenvironment in vivo in vitro, and further, the culture cost and the operation difficulty are increased.

Three-dimensional (3D) bioprinting is based on computer-aided additive manufacturing technology, distribution and combination of biomaterials, cells, growth factors and the like in a 3D structure are accurately controlled, and a constructed model can simulate a three-dimensional microenvironment required by cells in vivo and promote interaction of the cells and the extracellular matrix, so that biological behaviors of the cells are better maintained.

Citation 1 discloses a method for inducing stem cell in vitro directional differentiation by non-contact co-culture, which comprises embedding induced cells with induced differentiation function in biological microcapsules, co-culturing the microcapsules with stem cells, and realizing the directional induced differentiation of the stem cells by the directional induction effect of the induced cells in the microcapsules. The stem cells co-cultured with the microcapsules are cultured in a conventional two-dimensional growth mode or in a three-dimensional growth mode in the biological scaffold material. The microcapsule membrane in the method can enable the induction factors secreted by the induced cells to act on the external stem cells through the microcapsules, and can also isolate the induced cells from the stem cells in an immune manner without direct contact, thereby being beneficial to the separation and harvesting of different cells.

Citation 2 discloses a magnetic separation type cell three-dimensional co-culture method, wherein induced cells and magnetic medium particles are embedded in sodium alginate microcapsules together, the microcapsules and calcium alginate micro-gel beads embedded with stem cells are subjected to non-contact three-dimensional co-culture in the same system, directional differentiation of the stem cells is regulated and controlled by soluble factors secreted by the induced cells in the microcapsules, after the co-culture is finished, the microcapsules and the micro-gel beads are separated by a magnetic field, and the micro-gel beads are dissolved by a sodium citrate solution, so that pure differentiated cells are further harvested. The microcapsule and the microcapsule bead of the method can provide an approximate in-vivo three-dimensional growth environment for cells, the microcapsule membrane can enable induction factors secreted by the induced cells to act on stem cells through the microcapsule, and meanwhile, the immune isolation of different types of cells is realized, and the magnetic medium particles and the magnetic separation device are favorable for the separation and the harvest of the induced cells and the differentiated cells.

The above cited documents achieve non-contact co-culture with target cells by encapsulating induced cells with microcapsules, and although the phenomenon of a nutrient concentration gradient in the Transwell cell co-culture technique is avoided, the microcapsules exist independently of each other in the co-culture system, and are discontinuous between the microcapsules, making it difficult to simulate a continuous tissue structure in a living body. In addition, the microballoons may sink to the bottom of the culture dish due to gravity, and it is difficult to control the uniformity of the co-culture system.

Cited documents:

cited document 1: CN102102090A

Cited document 2: CN103849593A

Disclosure of Invention

Problems to be solved by the invention

In view of the technical problems in the prior art, for example: the cell co-culture system constructed by the microspheres has the problems that the microcapsules are discontinuous and the continuous tissue structure in a living body is difficult to simulate. The invention aims to provide a cell co-culture system which has continuity and integrity of structure and can simulate the continuity structure of tissues in vivo to a certain extent.

Another object of the present invention is to provide a method for constructing a cell co-culture system, which is easy to obtain raw materials and simple and easy to perform preparation steps.

Another object of the present invention is to provide an application of a cell co-culture system.

Means for solving the problems

(1) A cell co-culture system, comprising:

a hydrogel scaffold containing first cultured cells therein;

a liquid matrix in which the hydrogel scaffold is disposed such that the first cultured cells are present in the liquid matrix, the liquid matrix containing the second cultured cells therein.

(2) The cell co-culture system according to (1), wherein the hydrogel scaffold is a three-dimensional scaffold; optionally, the hydrogel scaffold is a three-dimensional scaffold with a lattice structure; preferably, the hydrogel scaffold is obtained by means of three-dimensional bioprinting.

(3) The cell co-culture system of (1) or (2), wherein the hydrogel scaffold is formed from a hydrogel system, wherein the hydrogel system comprises a hydrogel material and first cultured cells;

preferably, the hydrogel system is subjected to three-dimensional bioprinting to form hydrogel fiber yarns, the volume filling rate of the hydrogel fiber yarns in the hydrogel support is 20% -40%, and the diameter of the hydrogel fiber yarns is 0.45-0.6 mm; preferably, the hydrogel material comprises one or a combination of more than two of sodium alginate, gelatin, collagen, chitosan and hyaluronic acid.

(4) The cell co-culture system according to any one of (1) to (3), wherein the hydrogel scaffold is obtained by crosslinking treatment; preferably, the hydrogel scaffold is obtained by subjecting a cross-linked precursor scaffold containing sodium alginate to the cross-linking treatment using a cross-linking agent containing calcium ions.

(5) The cell co-culture system according to any one of (1) to (4), wherein the liquid medium is a cell culture medium.

(6) The cell co-culture system according to any one of (1) to (5), wherein the first cultured cells are cells in which proliferation is inhibited in the hydrogel scaffold; preferably, the first cultured cell is a mesenchymal stem cell or an osteoblast cell;

the second cultured cells are cells whose proliferation is promoted in the liquid medium; preferably, the second cultured cell is a hematopoietic stem cell, NK cell, T cell or B cell.

(7) A method for constructing a cell co-culture system according to any one of (1) to (6), comprising the steps of:

the preparation method comprises the following steps: mixing the first cultured cell with a hydrogel material to form a hydrogel system;

a printing step: obtaining a cross-linked precursor scaffold by using the hydrogel system as biological ink and performing three-dimensional biological printing;

a crosslinking step: treating the cross-linked precursor scaffold with a cross-linking agent to promote the hydrogel system to form a cross-linked structure to obtain a hydrogel scaffold;

mixing: transferring the hydrogel scaffold into a liquid matrix, allowing the first cultured cells to be present in the liquid matrix, and inoculating the second cultured cells into the liquid matrix to obtain the cell co-culture system;

optionally, the construction method further comprises the following steps:

culturing the hydrogel scaffold: after the crosslinking step, the hydrogel scaffold is placed in a cell culture medium for culturing, and the hydrogel scaffold after culturing is transferred to a liquid matrix.

(8) The construction method according to (7), wherein the hydrogel material comprises gelatin and sodium alginate, and the cross-linking agent is CaCl₂A solution; preferably, the content of the first cultured cell in the hydrogel system is 1X 10⁶one/mL to 3X 10⁶The concentration of the gelatin is 5% (w/v) to 15% (w/v), and the concentration of the sodium alginate is 0.5% (w/v) to 1.5% (w/v); the CaCl is₂The concentration of the solution is 2% (w/v) to 3% (w/v), and the crosslinking time is 2 to 3 minutes; preferably, the amount of inoculation of said second cultured cells in said liquid matrix is 5000-20000 cells/mL.

(9) The building method according to (7) or (8), wherein in the printing step, the printing rate is 2mm/s to 10 mm/s.

(10) Use of a cell co-culture system constructed according to the cell co-culture system of any one of (1) to (6) or the construction method of any one of (7) to (9) for preparing a model for in vitro studies;

optionally, the in vitro study comprises a cell interaction study, a tissue formation study, and/or a drug screening study.

ADVANTAGEOUS EFFECTS OF INVENTION

In one embodiment of the present invention, the hydrogel scaffold in the cell co-culture system of the present invention can simulate the three-dimensional environment required for cell co-culture in vivo, and the scaffold has a stable structure and can support long-term co-culture. Compared with a microsphere co-culture system, the cell co-culture system has better structural continuity and integrity. The hydrogel support can be constructed into different shapes and wrapped by different types of first culture cells according to requirements, so that a customized co-culture model can be constructed conveniently.

In another embodiment of the present invention, the first cultured cell in the cell co-culture system of the present invention is a mesenchymal stem cell, and the second cultured cell is a hematopoietic stem cell, and the cell co-culture system of the present invention can promote the proliferation of the hematopoietic stem cell and maintain the biological behavior of the hematopoietic stem cell, such as homing ability, etc.

In another embodiment of the invention, the method for constructing the cell co-culture system has the advantages of easily obtained raw materials, simple and feasible preparation process and good adaptability, and is suitable for constructing the cell co-culture system with a continuous three-dimensional structure.

Drawings

FIG. 1 shows a comparison of a three-dimensional bioprinted hydrogel scaffold with a lattice structure of the present invention with a biomimetic bone structure;

FIG. 2 shows a schematic diagram of the three-dimensional bioprinting and cross-linking principles and steps of the present invention;

FIG. 3 shows a scanning electron micrograph of a three-dimensional bioprinted hydrogel scaffold of the present invention;

FIG. 4 shows the distribution of first cultured cells in a three-dimensional bioprinted hydrogel scaffold containing the first cultured cells of the present invention;

FIG. 5 shows a scanning electron micrograph of a three-dimensional bioprinted cell co-culture system of the present invention after 10 days in vitro culture;

FIG. 6 is a photograph showing the distribution of first cultured cells in the cell co-culture system using two-dimensional culture according to the present invention;

FIG. 7 is a graph showing the proliferation of first cultured cells in a three-dimensional bioprinted cell co-culture system of the present invention compared to a two-dimensional cultured cell co-culture system; in FIG. 7, the abscissa represents the number of days of examination, and the ordinate is the value of each examination result relative to the examination result on the first day, and the higher the ordinate, the higher the proliferation degree; 2D represents a two-dimensional cell co-culture system, and 3D represents a three-dimensional cell co-culture system;

FIG. 8 is a graph showing the proliferation of second cultured cells in a three-dimensional bioprinted cell co-culture system of the present invention compared to a two-dimensional cultured cell co-culture system; the abscissa is the number of days tested and the ordinate is the number of cells. 2D represents a two-dimensional cell co-culture system, and 3D represents a three-dimensional cell co-culture system;

FIG. 9 is a graph showing the proliferation of a second cultured cell with a different initial population in a three-dimensional bioprinted cell co-culture system of the present invention compared to a two-dimensional cultured cell co-culture system; 2D represents a two-dimensional cell co-culture system, and 3D represents a three-dimensional cell co-culture system;

FIG. 10 is a graph showing a comparison of the proliferation of second cultured cells in a three-dimensional bioprinted cell co-culture system of the present invention, a two-dimensional cultured cell co-culture system, and a microspheroidal cell co-culture system; the abscissa is the number of days tested and the ordinate is the number of cells. 2D represents a two-dimensional cell co-culture system and 3D represents a three-dimensional cell co-culture system.

Detailed Description

Other objects, features and advantages of the present application will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.

It should be noted that:

in the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In this specification, the term "w/v" denotes the ratio of the mass of solute in solution to the volume of solution. Wherein the unit of solute mass is g, and the unit of solution volume is mL.

In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not. In this specification, the term "three-dimensional" has the same meaning as "3D", and three-dimensional means a spatial system formed by adding a direction vector to a planar two-dimensional system.

In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, means that a particular element (e.g., feature, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

< first aspect >

The present invention provides in < first aspect > a cell co-culture system comprising:

a hydrogel scaffold containing first cultured cells therein;

The hydrogel scaffold in the cell co-culture system can simulate the in-vivo three-dimensional environment required by cell co-culture, and the scaffold has stable structure and can support long-time co-culture. Compared with a microsphere co-culture system, the cell co-culture system has better structural continuity and integrity. The hydrogel support can be constructed into different shapes and wrapped by different types of first culture cells according to requirements, so that a customized co-culture model can be constructed conveniently. During co-culture, the first cultured cells remain in the three-dimensional microenvironment of the hydrogel scaffold, are uniformly distributed, and the secreted substances can pass through the hydrogel voids and be distributed in the liquid matrix, so that the second cultured cells can uniformly interact with the first cultured cells and the secreted substances.

Specifically, the cell co-culture system comprises:

hydrogel scaffold

In the present invention, the hydrogel scaffold is a three-dimensional scaffold that wraps the first cultured cell. The inventor finds in research that by adopting the hydrogel scaffold to construct the cell co-culture system, the three-dimensional continuous structure of the hydrogel scaffold can simulate the three-dimensional environment in an organism, and the wrapped first cultured cells are continuously and uniformly distributed in the hydrogel scaffold and cannot sink to the bottom of the liquid matrix, and the first cultured cells are distributed in the hydrogel scaffold in a three-dimensional space, so that the material communication between the first cultured cells and the second cultured cells is facilitated.

In particular, the hydrogel scaffold is formed from a hydrogel system, wherein the hydrogel system comprises a hydrogel material and first cultured cells. The hydrogel system in the present invention refers to a type of matrix that is extremely hydrophilic, crosslinked in its internal structure, or capable of forming a crosslinked structure under the action of a crosslinking agent, which is capable of providing environmental conditions for the growth of the first cultured cells. The hydrogel system has biophysical properties very similar to those of natural tissues and can serve as a substrate for the growth of first cultured cells.

In the present invention, the first cultured cells are cells in which proliferation is inhibited in the hydrogel scaffold. The first culture cells are wrapped in the hydrogel support, the hydrogel support can effectively limit the expansion of feeder layer cells, and the survival state and function of the first culture cells can be guaranteed in the hydrogel support. The secretion of the first cultured cell can enter the liquid matrix through the hydrogel support and interact with the second cultured cell in the liquid matrix, so that the proliferation of the second cultured cell is promoted, and the function of a feeder layer cell is realized. Compared with the traditional feeder layer cells, the first cultured cells do not need to be subjected to ultraviolet irradiation treatment or drug treatment to inhibit the cell proliferation capacity, so that the first cultured cells are prevented from generating genetic variation, or the co-culture of the second cultured cells is prevented from being influenced by adding drugs.

Specifically, the first cultured cell is a mesenchymal stem cell or an osteoblast cell.

In the present invention, the term "mesenchymal stem cells" (MSCs) is a pluripotent stem cell having all the commonalities of stem cells, i.e., self-renewal and multipotentiality. Preferably, the mesenchymal stem cell of the present invention is a human mesenchymal stem cell. Human mesenchymal stem cells are obtained from the human body and have been known to promote the proliferation of hematopoietic stem cells in vitro. And the stability of the human mesenchymal stem cells is good, the variation is not easy to occur in the in vitro culture process, and the variation of the second cells can not be caused.

In the present invention, the term "osteoblasts" (OBs) are mainly differentiated from mesenchymal progenitor cells within the stroma of the inner and outer periosteum and bone marrow, and osteoblasts have been confirmed to promote the proliferation of the second cultured cell as the first cultured cell.

In the present invention, the hydrogel material may be various types of materials commonly used in the art for forming hydrogels, and the present invention is not particularly limited thereto. In some preferred embodiments, the hydrogel material comprises one or a combination of two or more of sodium alginate, gelatin, collagen, chitosan, hyaluronic acid. The hydrogel material has good biocompatibility, and can realize the culture of cells and the maintenance of cell functions; the hydrogel material forms a hydrogel scaffold after crosslinking, and has better support for the first cultured cells, so that the first cultured cells are uniformly distributed in the hydrogel scaffold.

In some embodiments, the hydrogel system is bioprinted three-dimensionally to form hydrogel filaments. The hydrogel fiber silk is formed by three-dimensional biological printing, and is suitable for constructing hydrogel supports in various shapes and structures, so that individual accurate control of a three-dimensional microenvironment is realized.

Further, the hydrogel fiber has a volume filling rate of 20% to 40% in the hydrogel scaffold, for example, the volume filling rate of the hydrogel fiber may be 20%, 25%, 30%, 35%, 40%, etc. When the volume filling rate of the hydrogel fiber filaments is more than 40%, the printed pore structure of the hydrogel scaffold is unclear, and the fusion of the fiber filaments may occur. When the volume filling rate of the hydrogel fiber is less than 20%, the porosity of the hydrogel scaffold is too small, the pore diameter of the in vivo environment structure of the hematopoietic stem cells is about 100-300 μm, when the porosity is too small, the pore diameter of the hydrogel scaffold is far larger than the actual pore diameter of the in vivo structure, the preparation of a bionic structure is not facilitated, and in addition, the support performance of the scaffold is reduced due to the reduction of the porosity.

In some specific embodiments, the hydrogel scaffold is a three-dimensional scaffold structure including, but not limited to, a cylinder, a triangular prism, a cuboid, a pentagonal prism, a polygonal body, and the like. Optionally, the hydrogel scaffold has an edge length or diameter of 10mm to 20 mm. For example, the hydrogel scaffold may have sides or diameters of 12mm, 14mm, 16mm, 18mm, and the like. Optionally, the hydrogel scaffold has an edge length or diameter of 12 mm. Optionally, the thickness of the hydrogel scaffold is 1 mm-5 mm. For example, the hydrogel scaffold can have a thickness of 1mm, 2mm, 3mm, 4mm, 5mm, and the like. Preferably, the hydrogel scaffold has a thickness of 2.1 mm.

In some preferred embodiments, the hydrogel scaffold is a three-dimensional scaffold having a lattice structure. The hydrogel scaffold has a three-dimensional porous structure, so that the first cultured cells can absorb nutrient substances and exchange metabolic waste, secretion exchange between the first cultured cells and the second cultured cells is promoted, and a three-dimensional microenvironment closer to the in-vivo environment is established, so that biological processes such as interaction between cells in a normal physiological state are clarified. The three-dimensional porous structure of the hydrogel scaffold can also provide certain physical space support for the second cultured cells, so that the first cultured cells and the second cultured cells form an interwoven three-dimensional biological environment through a grid structure, and the real living environment of the cells is simulated to the maximum extent. Fig. 1 shows a comparison of a biomimetic bone structure with a hydrogel scaffold structure of the present invention, as shown in fig. 1, the biomimetic bone structure has a porous sponge structure, the hydrogel scaffold structure with a lattice structure of the present invention can well simulate the three-dimensional continuity structure, and a microsphere co-culture system cannot provide the structure for co-cultured cells.

In some specific embodiments, the hydrogel scaffold is cross-linked; preferably, the hydrogel scaffold containing sodium alginate is subjected to the cross-linking treatment using a cross-linking agent containing calcium ions. For example: and performing crosslinking treatment by using a calcium chloride solution. The sodium alginate can be converted into a stable hydrogel state through crosslinking treatment, so that effective support for the first cultured cell is formed, and a continuous and uniform structure close to a microenvironment in a living body is obtained.

Liquid matrix

In the present invention, the hydrogel scaffold is placed in the liquid matrix so that the first cultured cells are present in the liquid matrix, and the second cultured cells are contained in the liquid matrix. The liquid matrix provides a liquid environment for the growth and proliferation of the first cultured cells and the second cultured cells, and the first cultured cells and the second cultured cells realize communication between the cells by utilizing the liquid matrix to transport secretion substances of the respective cells, so that the cells have an integral uniform environment.

Specifically, the liquid matrix is a cell culture medium, which can supply nutrition to cells and basic substances for promoting cell reproduction and proliferation, and can also provide living environment for the growth and proliferation of cultured cells. Preferably, the liquid medium is a cell culture medium dedicated to the second cultured cells. The use of the cell culture medium dedicated to the second cells can provide a living environment favorable for the proliferation and growth of the second cultured cells while ensuring the function of the first cultured cells.

In the present invention, the second cultured cells are cells whose proliferation is promoted in the liquid medium. The hydrogel bracket is arranged in the liquid matrix, can provide support on a physical space for the second cultured cell and provides an effective three-dimensional space for the proliferation and the growth of the second cultured cell; meanwhile, the hydrogel scaffold can also inhibit the proliferation of the first cultured cells and reduce the consumption of nutrient substances in the liquid matrix so as to promote the proliferation of the second cultured cells. The secretory substance of the first cultured cell acts on the second cultured cell, and the proliferation of the second cultured cell is also promoted.

Specifically, the second cultured cell is a hematopoietic stem cell, an NK cell, a T cell, or a B cell.

In the present invention, the term "Hematopoietic stem cells" (HSCs) are adult stem cells in the blood system, a heterogeneous population with the ability to self-renew for a long period of time and the potential to differentiate into various types of mature blood cells. The source of the hematopoietic stem cells of the present invention is not particularly limited, and may be human or murine. Preferably, human hematopoietic stem cells are selected as the second cultured cells, and the surface marker of human hematopoietic stem cells which are widely used at present is LineageCD34CD 38.

The term "NK cell" refers to a natural killer cell (NK), an immune cell of major importance to the body.

The term "T cell" is also called T lymphocyte (T lymphocyte), and is a bone marrow-derived lymphoid stem cell that, after differentiation, developmental maturation in the thymus, distributes to immune organs and tissues throughout the body through lymph and blood circulation to exert immune function.

The term "B cell" is also called B lymphocyte (B lymphocyte), and is a pluripotent stem cell derived from bone marrow and developed by differentiation from hematopoietic stem cells in bone marrow.

In some specific embodiments, the first cultured cell is a mesenchymal stem cell, and the second cultured cell is a hematopoietic stem cell, and the cell co-culture system of the present invention can promote the proliferation of the hematopoietic stem cell and maintain the biological behavior of the hematopoietic stem cell, such as homing ability, etc.

According to the cell co-culture system, the hydrogel support wraps the first culture cell, the first culture cell and the second culture cell are co-cultured in the liquid matrix, when the second culture cell is collected, the second culture cell can be collected only by gently blowing and beating the support, the target cell does not need to be purified after collection, and the experimental efficiency can be effectively improved. Compared with the Transwell co-culture technology, the cell co-culture system provided by the invention can eliminate the concentration gradient phenomenon of nutrient substances and reduce the culture cost. Compared with a microsphere co-culture system, the cell co-culture system can simulate a three-dimensional continuous structure in vivo and provide a three-dimensional microenvironment closer to that in an organism.

< second aspect >

The < second aspect > of the present invention provides a method for constructing a cell co-culture system according to the < first aspect > of the present invention, comprising the steps of:

a crosslinking step: treating the cross-linked precursor scaffold with a cross-linking agent to promote the hydrogel system to form a cross-linked structure to obtain a hydrogel scaffold; mixing: transferring the hydrogel scaffold into a liquid matrix, allowing the first cultured cells to be present in the liquid matrix, and inoculating the second cultured cells into the liquid matrix to obtain the cell co-culture system;

optionally, the construction method further comprises the following steps:

The construction method of the cell co-culture system has the advantages of easily obtained raw materials, simple and easy preparation process and good adaptability, and is suitable for constructing the cell co-culture system with a continuous three-dimensional structure.

Illustratively, the specific operation of the steps employed in the present invention is as follows:

raw Material preparation step

In the present invention, the raw material preparation step includes mixing the first cultured cell with a hydrogel material to form a hydrogel system, and using the hydrogel system as a bio-ink for three-dimensional bio-printing.

In some embodiments, the hydrogel material may be a material commonly used in the art for forming hydrogels, including one or a combination of two or more of sodium alginate, gelatin, collagen, chitosan, hyaluronic acid. In some embodiments, the hydrogel material comprises gelatin and sodium alginate. The hydrogel scaffold is prepared from gelatin and sodium alginate hydrogel materials, and is suitable for constructing hydrogel scaffolds with good biocompatibility and continuous structures. Preferably, the gelatin is present at a concentration of 5% (w/v) to 15% (w/v), for example, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, etc.; the concentration of sodium alginate is 0.5% (w/v) to 1.5% (w/v), for example, the concentration of sodium alginate is 0.5%, 0.8%, 1%, 1.2%, 1.5%, etc. When the concentration of the gelatin is 5% (w/v) to 15% (w/v), and the concentration of the sodium alginate is 0.5% (w/v) to 1.5% (w/v), the three-dimensional network polymer with a stable gel state can be printed and crosslinked. Wherein, the concentration of the gelatin and the concentration of the sodium alginate are both the final concentration of the gelatin and the sodium alginate in the hydrogel system.

In a preferred embodiment, 15% (w/v) gelatin and 4% (w/v) sodium alginate are mixed with the first cultured cells in a volume ratio of 2:1:1 to obtain a hydrogel system having a final concentration of 7.5% (w/v) gelatin and a final concentration of 1% (w/v) sodium alginate.

In some embodiments, the hydrogel system comprises 1 × 10 cells of the first culture⁶one/mL to 3X 10⁶One cell/mL, for example, the content of the first cultured cells is 1X 10⁶1.5X 10 units/mL⁶2X 10 units/mL⁶2.5X 10 units/mL⁶3X 10 pieces/mL⁶one/mL, etc. When the content of the first cultured cells is 1X 10⁶one/mL to 3X 10⁶Per mL, when the content of the first cultured cells is less than 1X 10⁶At one/mL, the cell hydrogel structure printed has a low cell density, and insufficient communication between the first cultured cells affects the cell state and reduces its function. When the content of the first cultured cells is higher than 3X 10⁶At a cell/mL, the cell density is too high, which may inhibit contact of the cells, and affect the state of the first cultured cells, thereby causing differentiation.

Printing step

In the invention, the hydrogel system is used as biological ink, and the hydrogel scaffold is obtained through three-dimensional biological printing. After the hydrogel system forms a hydrogel scaffold in a three-dimensional biological printing mode, the first cultured cells grow in a three-dimensional space to form a continuous structure. The three-dimensional biological printing mode can adopt a three-dimensional single-nozzle biological printer, a three-dimensional double-nozzle biological printer and a three-dimensional multi-nozzle biological printer for printing. The diameter of the bioprinter nozzle is 0.2 mm-0.4 mm, and the bioprinter nozzle with the diameter of 0.2 mm-0.4 mm is selected to be printable to obtain the hydrogel fiber yarn with the diameter of 0.45-0.6 mm. For example, the bioprinter nozzle has a diameter of 0.21mm, 0.23mm, 0.26mm, 0.31mm, 0.33mm, and the like. Preferably, the diameter of the bioprinter nozzle is 0.26 mm. When the diameter of the nozzle of the bioprinter is less than 0.2mm, the shearing force of the cells passing through the nozzle may be excessive, resulting in a low survival rate of the cells during printing. When the diameter of a nozzle of the bioprinter is larger than 0.4mm, the hydrogel fiber yarns printed by the bioprinter are increased, so that the printing resolution is reduced, the increased yarn diameter of the hydrogel fiber yarns influences the exchange efficiency of first cultured cells in hydrogel and external substances, and the survival rate of the cells is reduced.

Furthermore, the diameter of the hydrogel fiber silk printed by the nozzle of the biological printer is 0.45-0.6 mm, for example, the diameter of the hydrogel fiber silk is 0.45mm, 0.52mm, 0.55mm, 0.57mm, 0.6mm, etc.

In some specific embodiments, the temperature of the bioprinter nozzle is controlled to be 20-30 ℃; for example, the temperature of the bioprinter nozzle is controlled at 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃ and the like; preferably, the temperature of the bioprinter nozzle is controlled to be 25 ℃.

In some embodiments, in the printing step, the shape and size of the hydrogel scaffold and the printing speed of the printer may be preset using computer software.

In some specific embodiments, in the step of printing, the printing speed is 2mm/s to 10 mm/s; for example, the printing speed is 3mm/s, 4mm/s, 5mm/s, 6mm/s, 7mm/s, 8mm/s, 9mm/s, or the like. Preferably, the printing speed is 5 mm/s.

Step of crosslinking

In the present invention, after the printing step, a crosslinking agent is used to promote the hydrogel system to form a crosslinked structure; the cross-linking treatment can convert sodium alginate into a stable hydrogel state, and the cross-linking treatment can enhance the mechanical strength of the hydrogel, provide wrapping support for the first cultured cell and provide an adhesive three-dimensional structure for the second cultured cell.

In some embodiments, the crosslinking agent is CaCl₂A solution; hydrogel system in CaCl₂The time of the cross-linking treatment in the solution is 2-3 min. Preferably, the CaCl₂The concentration of the solution is 2% (w/v) to 3% (w/v).

In some embodiments, after the crosslinking treatment of the hydrogel scaffold with the crosslinking agent, the hydrogel scaffold is washed with a buffer to remove residual crosslinking agent. Optionally, the hydrogel scaffold is washed with PBS buffer.

Mixing step

In the present invention, the hydrogel scaffold is transferred to a liquid medium, the first cultured cells are allowed to exist in the liquid medium, and the second cultured cells are seeded into the liquid medium to obtain the cell co-culture system. After the second cultured cells are mixed with the hydrogel scaffold in the liquid matrix, the second cultured cells can be clustered and adhered to the surface of the hydrogel scaffold, which shows that the hydrogel scaffold constructed by the 3D bioprinting technology is beneficial to the adhesion and growth of the second cultured cells.

In some embodiments, the second cultured cell is resuspended in a cell culture medium, and the hydrogel scaffold containing the first cultured cell is then placed in the cell culture medium to complete the construction of the three-dimensional cell culture system. Preferably, the cell culture medium is a medium dedicated to the second culture of cells.

Culture step of hydrogel scaffold

In the present invention, after the crosslinking step, the hydrogel scaffold is placed in a cell culture medium for culturing, and the hydrogel scaffold after culturing is transferred to a liquid medium. The activity of the first cultured cells within the hydraulic scaffold may be maintained by the culturing step, facilitating interaction between the first cultured cells and the second cultured cells.

In some embodiments, the hydrogel scaffold is placed in fresh medium and then incubated at 37 ℃ with 5% CO₂Cultured in a cell culture box.

< third aspect >

The invention provides in < a third aspect > use of a cell co-culture system as described in < the first aspect >, or a method of construction as described in < the second aspect >, for the preparation of a model for in vitro studies, optionally including cell interaction studies, tissue formation studies, and/or drug screening studies.

The cell co-culture system constructed by the invention can simulate the in-vivo environment to a great extent, observe the interaction between cells, make up the defect that the interaction between cells cannot be reflected in monolayer cell culture and the defect that the microsphere technology cannot form a continuous structure, and has wide prospects in the research and practical application in the fields of life science, medicine and pharmacy.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In the present invention, the manufacturers, models or sources of the materials and instruments used are as follows:

mesenchymal stem cells, purchased from Sciencell (CA, USA);

hematopoietic stem cells, purchased from Beijing Nuo as an organism;

the special cell culture medium for the hematopoietic stem cells comprises the following components: StemBan^TMSFEM II medium(STEMCELL,Vancouver,Canada)，50ng/mL recombinant human stem cell factor(SCF,proteintech,Chicago,US),50ng/mL recombinant human thrombopoietin(TPO,proteintech,Chicago,US)，50ng/mL recombinant human Flt3-Ligand(FLT-3,proteintech,Chicago,US)；

Fetal bovine serum, purchased from Gibco;

DMEM high-glucose medium, purchased from Gibco, Grand Island, NY, USA;

sodium alginate (S100128) and gelatin (G108396) were purchased from alatin corporation (shanghai, china);

the alamar blue kit was purchased from YEASEN (shanghai, china);

biological 3D Printer (

3D bio-printer NORM series)；

Cell counter (counttarrigel s2, counttstar, shanghai, china).

Example 1: cell co-culture system for three-dimensional biological printing

The embodiment constructs a cell co-culture system of mesenchymal stem cells and hematopoietic stem cells, and the specific construction steps are as follows:

1. planting human umbilical cord blood-derived mesenchymal stem cells in a 10cm culture dish, placing at 37 deg.C and 5% CO₂Culturing in an incubator, wherein the culture medium is a DMEM high-sugar medium containing 10% fetal calf serum, the culture medium is replaced every 2-3 days, and when the cell growth rate is 80-85%, the cells are digested by pancreatin (Gibco) and collected by conventional centrifugation for later use.

2. Sodium alginate and gelatin are used as hydrogel materials, and human MSC cells are used as first cultured cells. Preparing sodium alginate solution with mass volume fraction of 4% and gelatin solution with mass volume fraction of 15%, and sterilizing at high temperature under high pressure. Firstly 4 is multiplied by 10⁶the/mL MSC cells were resuspended in 1mL of medium and mixed with 4% sodium alginate and 15% gelatin solution at a volume ratio of 1:1:2 to give final concentrations of 7.5% gelatin, 1% sodium alginate and 1X 10⁶Generation of/mL MSC cellsAnd (4) ink. The bio-ink is a hydrogel system comprising a hydrogel material and a first cultured cell.

3. Three-dimensional printing was performed by the three-dimensional bioprinting and cross-linking principle and step scheme of the present invention as shown in figure 2. Specifically, during printing, the prepared hydrogel system is filled into a 1mL injector as a printing material and is placed in a pushing clamping groove of a printer and connected with a printing nozzle. The computer software presets the side length of the printed hydrogel support to be 12mm, the thickness to be 2.1mm and the filling rate to be 30%. The square grid support was printed using a biological 3D printer. The temperatures of the nozzle and the printing chamber were controlled at 25 ℃ and 8 ℃ respectively. The diameter of the nozzle is selected to be 0.26mm, the printing speed is controlled to be 5mm/s, and after the printing is finished, the cross-linking precursor bracket loaded with the mesenchymal stem cells is immersed into a 3% (W/V) calcium chloride solution for 3 minutes to cross-link the sodium alginate. The scaffolds were then gently washed three times with phosphate buffer solution to remove excess cross-linking agent prior to incubation. Adding fresh culture medium, standing at 37 deg.C and 5% CO₂Culturing in an incubator.

4. Taking human hematopoietic stem cells as second cultured cells, removing the original culture medium of the cell hydrogel scaffold after 24 hours, and adding 2ml of special culture medium for human umbilical cord blood-derived hematopoietic stem cells again. Then, the hematopoietic stem cells were recovered from the liquid nitrogen, resuspended in a hematopoietic stem cell-dedicated medium, and the hematopoietic stem cells were seeded in a culture dish containing a hydrogel scaffold of mesenchymal stem cells in a cell count of 20000 per well (the content of hematopoietic stem cells was 10000 cells/mL), thereby completing the construction of a three-dimensional cell co-culture system.

5. The culture medium was changed every 3 days, as follows: collecting cell suspension, centrifuging, removing old culture medium to obtain expanded hematopoietic stem cells, adding 2ml fresh hematopoietic stem cell culture medium to resuspend cells, and adding into original culture well again for continuous culture. The medium exchange is completed.

The experimental results are as follows:

the hydrogel scaffold formed by printing is shown in fig. 3, and the hydrogel scaffold has a grid structure communicated with the inside so as to facilitate the exchange of nutrients and metabolic wastes and facilitate the interaction between mesenchymal stem cells and hematopoietic stem cells.

The spatial distribution of the first cultured cells in the hydrogel scaffold is shown in FIG. 4, and the first cultured cells are in a three-dimensional space part in the hydrogel scaffold, which is beneficial to promote cell-to-cell communication.

The scanning electron micrograph of the cell co-culture system after 10 days of in vitro culture is shown in FIG. 5. As can be seen from FIG. 5, under the three-dimensional co-culture system, the second cultured cells adhered to the surface of the hydrogel scaffold in a cluster, indicating that the feeder cells hydrogel scaffold constructed by the biological 3D printing technology is helpful for the adhesion and growth of the second cultured cells.

Example 2

This example constructed a cell co-culture system according to the method provided in example 1, differing from the cell co-culture system constructed in example 1 only in that: in step 4, the number of hematopoietic stem cells planted was 10000 per well (the hematopoietic stem cell content was 5000 per mL).

Example 3

This example constructed a cell co-culture system according to the method provided in example 1, differing from the cell co-culture system constructed in example 1 only in that: in step 4, the number of hematopoietic stem cells planted was 30000 per well (the hematopoietic stem cell content was 15000 per mL).

Example 4

This example constructed a cell co-culture system according to the method provided in example 1, differing from the cell co-culture system constructed in example 1 only in that: in step 4, the number of hematopoietic stem cells planted was 40000 per well (the hematopoietic stem cell content was 20000 per mL).

Comparative example 1: two-dimensional cell co-culture system

1. Firstly, culturing mesenchymal stem cells in vitro by adopting a culture dish, digesting and collecting cells by conventional centrifugation when the cell growth rate is 80-85%, resuspending by a DMEM high-sugar culture medium containing 10% fetal calf serum, planting the cells in a 24-well plate according to the number of the cells of 150000 per well, placing the 24-well plate in the culture dish at 37 ℃ and 5% CO₂Culturing in an incubator.

2. After 24 hours, the original medium was removed, 2mL of hematopoietic stem cell medium was newly added, and the revived primary hematopoietic stem cells were seeded into mesenchymal stem cell-containing wells (hematopoietic stem cell content: 10000 cells/mL) in an initial cell count of 20000 per well, respectively.

The experimental results are as follows:

as shown in fig. 6, the first cultured cells grew as a monolayer, and were deficient in cell-cell and cell-extracellular matrix interactions, and were unable to mimic the three-dimensional biological structure of the in vivo microenvironment.

Comparative example 2

This comparative example a 2D cell co-culture system was constructed according to the method of comparative example 1, differing from comparative example 1 only in that the hematopoietic stem cells were planted in an amount of 10000 cells/well (the content of hematopoietic stem cells was 5000 cells/mL) in step 2.

Comparative example 3

This comparative example a 2D cell co-culture system was constructed according to the method of comparative example 1, differing from comparative example 1 only in that the hematopoietic stem cell engraftment was 30000 cells/well (the hematopoietic stem cell content was 15000 cells/mL) in step 2.

Comparative example 4

This comparative example a 2D cell co-culture system was constructed according to the method of comparative example 1, differing from comparative example 1 only in that the hematopoietic stem cell engraftment was 40000 cells/well (hematopoietic stem cell content was 20000 cells/mL) in step 2.

Comparative example 5: construction of three-dimensional cell co-culture system by microsphere technology

0.30ml of mesenchymal stem cell suspension containing 1% (W/V) sodium alginate is prepared, wherein the number of cells is 150000. The microspheres were formed by dropping the weight of the droplets into a 3% (W/V) calcium chloride solution using a minipump control.

Filtering the calcium chloride solution to collect microspheres, washing with normal saline, adding into a culture dish, and adding fresh hematopoietic stem cell culture medium.

20000 hematopoietic stem cells were added to the culture dish containing microspheres to complete the construction of a microsphere three-dimensional co-culture system (the content of hematopoietic stem cells was 10000 cells/mL).

And (3) experimental verification:

1. when the cell states of the cells in the example 1 and the comparative example 1 are detected by using an alamar blue kit respectively in the cell co-culture system for 1 day, 3 days, 5 days, 8 days and 16 days, the proliferation condition of the mesenchymal stem cells of the first culture cell is counted, and the result is shown in figure 7

2. The proliferation of hematopoietic stem cells as second cultured cells in example 1 and comparative example 1 was counted using a cell counter at 0, 4 and 10 days of culture in the cell co-culture system. As a result, hematopoietic stem cells (CD 34) in a three-dimensional co-culture system are shown in FIG. 8⁺CD38^-Cells) exhibited a higher expansion number, indicating that the cell co-culture system can promote the proliferation of the second cultured cells while inhibiting the proliferation of the first cultured cells.

3. After 10 days of culture in the cell co-culture system, the second cultured cells (hematopoietic stem cells, CD 34) in examples 2, 3 and 4 and comparative examples 2, 3 and 4 were counted using a cell counter⁺CD38^-) Fold amplification of (4). The results are shown in FIG. 9: the three-dimensional cell co-culture systems in examples 2, 3 and 4 all showed higher expansion fold than the two-dimensional cell co-culture system, indicating that the three-dimensional co-culture system has advantages for hematopoietic stem cell expansion.

4. When the cell co-culture system was cultured for 7 days, the proliferation of hematopoietic stem cells as the second cultured cells in example 1, comparative example 1 and comparative example 5 was counted using a cell counter. As a result, as shown in FIG. 10, hematopoietic stem cells (CD 34) in a three-dimensional co-culture system constructed based on the three-dimensional bioprinting technique in example 1⁺CD38^-Cells) show higher amplification quantity, which shows that the three-dimensional culture environment constructed by the invention has better continuity and structural integrity in structure than the existing microsphere co-culture environment, and the three-dimensional culture environment has better continuity and structural integrity on the body of hematopoietic stem cellsThe external amplification has a more positive promoting effect.

The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A cell co-culture system, comprising:

a hydrogel scaffold containing first cultured cells therein;

2. The cell co-culture system according to claim 1, wherein the hydrogel scaffold is a three-dimensional scaffold; optionally, the hydrogel scaffold is a three-dimensional scaffold with a lattice structure; preferably, the hydrogel scaffold is obtained by means of three-dimensional bioprinting.

3. A cell co-culture system according to claim 1 or 2, wherein the hydrogel scaffold is formed from a hydrogel system, wherein the hydrogel system comprises a hydrogel material and first cultured cells;

4. A cell co-culture system according to any one of claims 1 to 3, wherein the hydrogel scaffold is obtained by cross-linking treatment; preferably, the hydrogel scaffold is obtained by subjecting a cross-linked precursor scaffold containing sodium alginate to the cross-linking treatment using a cross-linking agent containing calcium ions.

5. A cell co-culture system according to any of claims 1-4, wherein the liquid substrate is a cell culture medium.

6. The cell co-culture system according to any one of claims 1 to 5, wherein the first cultured cell is a cell in which proliferation is inhibited in the hydrogel scaffold; preferably, the first cultured cell is a mesenchymal stem cell or an osteoblast cell;

7. A method of constructing a cell co-culture system according to any one of claims 1 to 6, comprising the steps of:

optionally, the construction method further comprises the following steps:

8. The construction method according to claim 7, wherein the hydrogel material comprises gelatin and sodium alginate, and the cross-linking agent is CaCl₂A solution; preferably, the content of the first cultured cell in the hydrogel system is 1X 10⁶one/mL to 3X 10⁶The concentration of the gelatin is 5% (w/v) to 15% (w/v), and the concentration of the sodium alginate is 0.5% (w/v) to 1.5% (w/v); the CaCl is₂The concentration of the solution is 2% (w/v) to 3% (w/v), and the crosslinking time is 2 to 3 minutes; preferably, the amount of inoculation of said second cultured cells in said liquid matrix is 5000-20000 cells/mL.

9. The building method according to claim 7 or 8, wherein in the printing step, the printing rate is 2mm/s to 10 mm/s.

10. Use of a cell co-culture system according to any one of claims 1 to 6 or a cell co-culture system constructed according to the method of construction of any one of claims 7 to 9 for the preparation of a model for in vitro studies;