CN113416702B - Hematopoietic stem cell in-vitro 3D hydrogel culture system and construction method thereof - Google Patents
Hematopoietic stem cell in-vitro 3D hydrogel culture system and construction method thereof Download PDFInfo
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Abstract
The invention mainly aims to provide an in-vitro 3D hydrogel culture system for hematopoietic stem cells and a construction method thereof, wherein: the construction method of the hematopoietic stem cell in-vitro 3D hydrogel culture system comprises the following steps: obtaining primary marrow hematopoietic stem cells; resuspending the hematopoietic stem cells with an IMDM medium containing fetal bovine serum; uniformly mixing the type I collagen acidic solution, a 1N sodium hydroxide solution and 10xPBS, adding hematopoietic stem cells after the PH is regulated to be neutral, and uniformly mixing to form hydrogel; adding IMDM culture solution containing fetal bovine serum and cytokines into the hydrogel; preparing collagenase solution and sodium citrate buffer solution respectively, adding the sodium citrate buffer solution and the collagenase solution into the hydrogel respectively, dissolving the hydrogel and collecting cells. The invention has the advantages of good cell compatibility, proper mechanical strength, easy degradation and capability of maintaining the cell stem property and the marrow differentiation capability.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to an in-vitro 3D hydrogel culture system for hematopoietic stem cells and a construction method thereof.
Background
In the prior art, a common suspension culture system is adopted to perform in-vitro culture and directional regulation and control of the hematopoietic stem cells by adding different cytokine combinations, but the suspension culture system lacks mechanical support and cannot completely simulate the real in-vivo microenvironment of the hematopoietic stem cells;
Two-dimensional or three-dimensional nanofiber scaffolds prepared from synthetic polymer materials have been studied in part to provide a certain viscoelasticity, but can provide physical support for cell survival, and have been developed to some extent. However, due to the factors of high toxicity, poor biocompatibility, strong mechanical properties and the like of the high polymer material, the problems of unsatisfactory cell expansion or differentiation effect, difficulty in separation from the scaffold material, easiness in cell damage in the separation process and the like exist.
Disclosure of Invention
The invention mainly aims to provide an in-vitro 3D hydrogel culture system for hematopoietic stem cells and a construction method thereof, which are used for solving the problems in the prior art, wherein:
According to the embodiment of the invention, an in-vitro 3D hydrogel culture system for hematopoietic stem cells is provided, which comprises the following components: sodium alginate, type I collagen, IMDM culture medium, fetal bovine serum and growth factors; wherein, the sodium alginate is 0.05-0.5wt%, the collagen I is 1-3mg/mL, the IMDM culture medium is 10mg/mL, and the fetal bovine serum is 10v/v%.
Wherein the IMDM medium comprises: SCF 50ng/ml, flt3L 20ng/ml, TPO 20ng/ml.
The embodiment of the invention also provides a construction method of the hematopoietic stem cell in-vitro 3D hydrogel culture system, which comprises the following steps: obtaining primary marrow hematopoietic stem cells; resuspending the hematopoietic stem cells with an IMDM medium containing fetal bovine serum; uniformly mixing the type I collagen acidic solution, a 1N sodium hydroxide solution and 10xPBS, adding hematopoietic stem cells after the PH is regulated to be neutral, and uniformly mixing to form hydrogel; adding IMDM culture solution containing fetal bovine serum and cytokines into the hydrogel; preparing collagenase solution and sodium citrate buffer solution respectively, adding the sodium citrate buffer solution and the collagenase solution into the hydrogel respectively, dissolving the hydrogel and collecting cells.
Wherein the method further comprises: preparing sodium alginate solution, mixing the sodium alginate solution with I-type collagen acidic solution, 1N sodium hydroxide solution and 10 xPS uniformly, adding hematopoietic stem cells after PH is adjusted to be neutral, and mixing uniformly to form hydrogel; dropwise adding CaCl 2 solution on the surface of the hydrogel, and crosslinking CaCl 2 and sodium alginate to enhance the matrix rigidity of the hydrogel; the hydrogel was washed off with CaCl 2 solution.
Wherein, the step of preparing sodium alginate solution comprises: dissolving sodium alginate powder in sterile deionized water to obtain sodium alginate solution; and (3) carrying out high-temperature high-pressure sterilization treatment on the sodium alginate solution.
Wherein, the step of obtaining the primary marrow hematopoietic stem cells comprises: collecting bone marrow, and collecting to obtain marrow mononuclear cells by a density gradient centrifugation method; magnetic beads separate Lin - progenitor cells from bone marrow mononuclear cells; lin -CD117+ cells in bone marrow mononuclear cells were isolated by magnetic beads.
Wherein the IMDM medium comprises: the following cytokines: SCF, TPO, flt3L.
The hematopoietic stem cell in-vitro 3D hydrogel culture system has the advantages of good cell compatibility, proper mechanical strength, easy degradation and capability of maintaining the cell stem property and the myeloid differentiation capability.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application. In the drawings:
FIG. 1 is a flow chart of a method of constructing an in vitro 3D hydrogel culture system for hematopoietic stem cells according to an embodiment of the invention;
FIGS. 2A and 2B are schematic diagrams of cell compatibility assays of three-dimensional hydrogel culture systems constructed in accordance with embodiments of the present invention;
FIG. 3 is a schematic representation of the in vitro expansion effects of hematopoietic stem cells of a three-dimensional hydrogel culture system (3D) constructed in accordance with an embodiment of the present invention with two-dimensional horizontal culture (2D) and conventional fluid culture (NC);
FIGS. 4A and 4B are schematic diagrams of differentiation of a three-dimensional hydrogel culture system (3D) regulatable macrophage lineage constructed according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments of the present invention and corresponding drawings. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The following describes in detail the technical solutions provided by the embodiments of the present invention with reference to the accompanying drawings.
FIG. 1 is a flow chart of a method of constructing an in vitro 3D hydrogel culture system for hematopoietic stem cells according to an embodiment of the invention, the method comprising the steps of:
step S102, obtaining primary marrow hematopoietic stem cells;
step S104, adopting IMDM culture medium containing fetal bovine serum to resuspend hematopoietic stem cells;
Wherein, the fetal calf serum is 10v/v%, and the IMDM culture medium comprises SCF 50ng/ml, flt3L 20ng/ml and TPO 20ng/ml.
Step S106, uniformly mixing the type I collagen acidic solution, the 1N sodium hydroxide solution and the 10xPBS, adding hematopoietic stem cells after the PH is adjusted to be neutral, uniformly mixing and forming hydrogel;
Step S108, adding IMDM culture solution containing fetal bovine serum and cytokines into the hydrogel;
Step S110, respectively preparing collagenase solution and sodium citrate buffer, respectively adding the sodium citrate buffer and the collagenase solution into the hydrogel, dissolving the hydrogel and collecting cells.
In some embodiments of the present application, the hydrogel may further comprise sodium alginate solution, that is, the three-dimensional collagen hydrogel cell culture system specifically comprises the following components: sodium alginate, type I collagen, IMDM medium, fetal bovine serum, and growth factors. Wherein the contents of the components are as follows: sodium alginate 0.05-0.5wt%, collagen I1-3mg/mL, IMDM culture medium 10mg/mL, fetal bovine serum 10v/v%, SCF 50ng/mL, flt3L 20ng/mL, TPO 20ng/mL.
Then, caCl 2 solution is dripped on the surface of the hydrogel, and CaCl 2 and sodium alginate are crosslinked to enhance the matrix rigidity of the hydrogel. Next, after washing the hydrogel from CaCl 2 solution, IMDM medium containing fetal bovine serum and cytokines was added.
Embodiments of the present application are described in detail below with reference to the accompanying drawings.
The method for constructing the HSCs in-vitro 3D hydrogel culture system comprises the following steps of:
1. primary bone marrow hematopoietic stem cells were obtained.
1. Collecting bone marrow, collecting cells by density gradient centrifugation, adding PBS buffer solution precooled at 4 ℃ for washing for 2 times, and discarding the supernatant to obtain bone marrow mononuclear cells;
2. Lin - progenitor cells were sorted by magnetic beads.
(1) The immunomagnetic beads label the mononuclear cells.
Each 108 cells were resuspended with 400 μl of magnetic bead sorting buffer and 100 μl of biotin antibody mixture was added to label the cells; gently mixing, and incubating at 4deg.C for 10min; adding 300 μl of magnetic bead sorting buffer; 200 μl of avidin microbeads are added, mixed uniformly and incubated at 4deg.C for 15min; adding 10ml of magnetic bead sorting buffer solution, 300Xg,10min, and discarding the supernatant; adding 500ul buffer to resuspend cells;
(2) Negative cells were magnetically sorted.
The MAC medium-sized magnetic head is tightly attached to the middle position of the magnetic frame, and an LS sorting column is placed; fully infiltrating the sorting column with 3ml buffer solution; dripping the marked marrow mononuclear cell suspension into a separation column from a middle position at a constant speed, avoiding generating bubbles, and collecting Lin - cells from the lower part of the separation column; washing the sorting column 3 times with 3ml buffer; counting cells of the collected Lin - suspension;
3. Lin -CD117+ cells were sorted by magnetic beads.
(1) Immunomagnetic beads labeled Lin - cells.
Centrifuging the Lin - cell suspension collected in the step 2, 300g for 5min, and discarding the supernatant; mu.l of magnetic bead sorting buffer was added per 108 cells; 200 μl CD117 microbead labeled Lin - cells were added; gently mixing and placing the mixture in a place for incubation for 15min at 4 ℃; adding 10ml buffer solution, 300 Xg, 10min, and discarding supernatant; adding 500 μl buffer for use;
(2) Positive cells were magnetically sorted.
The MAC small-sized magnetic head is tightly attached to the middle position of the magnetic frame, and an MS sorting column is placed; fully infiltrating the sorting column with 500 μl buffer; dripping the Lin - cell suspension marked with the CD117 antibody into a separation column from the middle position at a constant speed to avoid generating bubbles; the column was washed 3 times with 500 μl buffer; removing the MS column combined with the positive cells from the magnetic field, and placing the MS column on a clean 15ml centrifuge tube; 1ml buffer solution is dripped into the center of the separation column at a constant speed, and positive cells with magnetic marks are pushed out by lightly pressurizing by a piston matched with an MS column; counting the collected Lin -CD117+ cells;
2. Three-dimensional hydrogel cell culture system preparation, wherein the system components include: IMDM medium, fetal bovine serum, collagen I, growth factors; the content of each component is as follows: collagen I1-3mg/mL, IMDM culture medium 10mg/mL, fetal bovine serum 10v/v%, SCF 50ng/mL, flt3L 20ng/mL, TPO 20ng/mL.
1. Preparing IMDM culture medium containing 10v/v% of fetal bovine serum, 50ng/ml of SCF, 20ng/ml of Flt3L and 20ng/ml of TPO, and re-suspending hematopoietic stem cells by using the freshly prepared culture medium;
2. the method comprises the steps of (1) pre-cooling an acidic solution of type I collagen, a 1N sodium hydroxide solution and 10xPBS, taking out the acidic solution, and placing the acidic solution on ice for operation;
3. Taking 1-3mg of collagen, rapidly and uniformly mixing, and pre-cooling on ice for 2min;
4. Precooling, taking out, and adjusting pH to neutrality with sodium hydroxide solution;
5. adding 100 μl of 10xPBS, rapidly mixing, and pre-cooling;
6. Adding 5x10 5 freshly extracted hematopoietic stem cells, and gently mixing;
7. adding sterile water to make up the volume of the system to 1ml;
8. Spreading the collagen-cell mixed solution obtained in the step 2 in a 12-hole plate, wherein 1 ml/hole is placed in a cell incubator at 37 ℃ for 30min;
9. After gel formation, the well plate was removed, IMDM medium containing cytokines (scf+tpo+flt3l) was added, and the well plate was placed in a 37 ℃ cell incubator for cell culture.
3. Degradation of collagen hydrogel systems and cell separation.
1. Preparing collagenase solution.
(1) Weighing 50-100mg collagenase powder, adding sterile deionized water to constant volume to 50ml, and preparing 1-2mg/ml collagenase solution;
(2) After the powder is completely dissolved, filtering and sterilizing by a disposable filter;
(3) Collecting with sterile centrifuge tube, packaging, and storing at-20deg.C;
2. digestion of hydrogels.
(1) Sucking the culture solution on the hydrogel, adding 1ml collagenase solution into each hole, and incubating for 1h at 37 ℃;
(2) After the collagen hydrogel was completely digested and dissolved, the cells were collected by centrifugation at 1000rpm for 5min with gentle shaking.
The construction method of the HSCs in vitro 3D hydrogel culture system according to the second embodiment of the application comprises the following steps:
1. Preparing sodium alginate solution.
1. Weighing 0.5-1g sodium alginate powder, slowly adding 250mL into a sterile conical flask under shaking, and continuously shaking until all sodium alginate powder is wetted and uniformly dispersed;
2. adding 80mL of sterile deionized water into a conical flask, stirring, and heating in a water bath kettle at 60 ℃ for 12h;
3. sterilizing at 120deg.C for 20min, cooling at room temperature, adding appropriate amount of sterile deionized water to volume of 100mL, cooling, and storing at 4deg.C in refrigerator.
2. Primary bone marrow hematopoietic stem cells were obtained.
1. Collecting bone marrow, collecting cells by density gradient centrifugation, adding PBS buffer solution precooled at 4 ℃ for washing for 2 times, and discarding the supernatant to obtain bone marrow mononuclear cells;
2. Lin - progenitor cells were sorted by magnetic beads.
(1) The immunomagnetic beads label the mononuclear cells.
Every 10 8 cells were resuspended with 400 μl of magnetic bead sorting buffer and 100 μl of biotin antibody mixture was added to label the cells; gently mixing, and incubating at 4deg.C for 10min; adding 300 μl of magnetic bead sorting buffer; 200 μl of avidin microbeads are added, mixed uniformly and incubated at 4deg.C for 15min; adding 10ml of magnetic bead sorting buffer solution, 300Xg,10min, and discarding the supernatant; cells were resuspended by adding 500ul buffer.
(2) Negative cells were magnetically sorted.
The MAC medium-sized magnetic head is tightly attached to the middle position of the magnetic frame, and an LS sorting column is placed; fully infiltrating the sorting column with 3ml buffer solution; dripping the marked marrow mononuclear cell suspension into a separation column from a middle position at a constant speed, avoiding generating bubbles, and collecting Lin - cells from the lower part of the separation column; washing the sorting column 3 times with 3ml buffer; cell counts were performed on the collected Lin - suspensions.
3. Lin -CD117+ cells were sorted by magnetic beads.
(1) Immunomagnetic beads labeled Lin - cells.
Centrifuging the Lin - cell suspension collected in the step 2, 300g for 5min, and discarding the supernatant; mu.l of magnetic bead sorting buffer was added per 10 8 cells; 200 μl CD117 microbead labeled Lin - cells were added; gently mixing and placing the mixture in a place for incubation for 15min at 4 ℃; adding 10ml buffer solution, 300 Xg, 10min, and discarding supernatant; add 500. Mu.l buffer for later use.
(2) Positive cells were magnetically sorted.
The MAC small-sized magnetic head is tightly attached to the middle position of the magnetic frame, and an MS sorting column is placed; fully infiltrating the sorting column with 500 μl buffer; dripping the Lin - cell suspension marked with the CD117 antibody into a separation column from the middle position at a constant speed to avoid generating bubbles; the column was washed 3 times with 500 μl buffer; removing the MS column combined with the positive cells from the magnetic field, and placing the MS column on a clean 15ml centrifuge tube; 1ml buffer solution is dripped into the center of the separation column at a constant speed, and positive cells with magnetic marks are pushed out by lightly pressurizing by a piston matched with an MS column; the collected Lin -CD117+ cells were subjected to cell counting.
3. Three-dimensional hydrogel cell culture system preparation, wherein the system components include: IMDM culture medium, fetal calf serum, sodium alginate, collagen I and growth factor; the content of each component is as follows: sodium alginate 0.05-0.1wt%, collagen I1-3mg/mL, IMDM culture medium solution 10mg/mL, fetal bovine serum 10v/v%, SCF 50ng/mL, flt3L 20ng/mL, TPO 20ng/mL.
1. Preparing IMDM culture medium containing 10v/v% of fetal bovine serum, 50ng/ml of SCF, 20ng/ml of Flt3L and 20ng/ml of TPO, and re-suspending hematopoietic stem cells by using the freshly prepared culture medium;
2. Precooling type I collagen acidic solution, 1N sodium hydroxide solution, 10xPBS, sodium alginate solution and 10-60mg/ml CaCl 2 solution, taking out, and placing on ice for operation;
3. Taking 1-3mg of collagen, adding 100 μl of sodium alginate solution, rapidly and uniformly mixing, and pre-cooling on ice for 2min;
4. Precooling, taking out, and adjusting pH to neutrality with sodium hydroxide solution;
5. adding 100 μl of 10xPBS, rapidly mixing, and pre-cooling;
6. Adding 5x10 5 freshly extracted hematopoietic stem cells, and gently mixing;
7. adding sterile water to make up the volume of the system to 1ml;
8. Spreading the collagen-cell mixed solution obtained in the step 2 in a 12-hole plate, wherein 1 ml/hole is placed in a cell incubator at 37 ℃ for 30min;
9. taking out after gel forming, uniformly dripping 1ml of 10-60mg/ml CaCl 2 onto the surface of the hydrogel, and placing the hydrogel in a cell incubator at 37 ℃ for crosslinking for 20min;
10. The well plate was removed, caCl 2 was aspirated, washed 3 times with hepes buffer, IMDM medium containing cytokines (SCF+TPO+Flt3L) was added, and the cells were cultured in a 37℃cell incubator.
4. Degradation of collagen hydrogel systems and cell separation.
1. Preparing collagenase solution.
Weighing 50-100mg collagenase powder, adding sterile deionized water to constant volume to 50ml, and preparing 1-2mg/ml collagenase solution; after the powder is completely dissolved, filtering and sterilizing by a disposable filter; collecting with sterile centrifuge tube, packaging, and storing at-20deg.C;
2. preparing 0.05-0.1mol/L sodium citrate buffer solution.
Weighing 4.17-8.34g of sodium citrate powder, and adding deionized water to a volume of 100ml; sterilizing at 120deg.C for 20min under high temperature and high pressure;
3. digestion of hydrogels.
Taking out a 12-well plate for hydrogel culture, discarding the culture solution of the supernatant, adding 1ml of sodium citrate buffer solution into each well, and incubating for 20min at 37 ℃; sucking the supernatant of the hydrogel, adding 1ml collagenase solution into each hole, and incubating for 1h at 37 ℃; after the collagen hydrogel was completely digested and dissolved, the cells were collected by centrifugation at 1000rpm for 5min with gentle shaking.
The method for constructing the HSCs in vitro 3D hydrogel culture system according to the third embodiment of the application comprises the following steps:
1. Preparing sodium alginate solution.
1. Weighing 2.5-5.0g of sodium alginate powder, slowly adding 250mL of sodium alginate powder into a sterile conical flask under shaking, and continuously shaking until all sodium alginate powder is wetted and uniformly dispersed;
2. adding 80mL of sterile deionized water into a conical flask, stirring, and heating in a water bath kettle at 60 ℃ for 12h;
3. sterilizing at 120deg.C for 20min, cooling at room temperature, adding appropriate amount of sterile deionized water to volume of 100mL, cooling, and storing at 4deg.C in refrigerator.
2. Primary bone marrow hematopoietic stem cells were obtained.
1. Collecting bone marrow, collecting cells by density gradient centrifugation, adding PBS buffer solution precooled at 4 ℃ for washing for 2 times, and discarding the supernatant to obtain bone marrow mononuclear cells;
2. Lin - progenitor cells were sorted by magnetic beads.
(1) The immunomagnetic beads label the mononuclear cells.
Every 10 8 cells were resuspended with 400 μl of magnetic bead sorting buffer and 100 μl of biotin antibody mixture was added to label the cells; gently mixing, and incubating at 4deg.C for 10min; adding 300 μl of magnetic bead sorting buffer; 200 μl of avidin microbeads are added, mixed uniformly and incubated at 4deg.C for 15min; adding 10ml of magnetic bead sorting buffer solution, 300Xg,10min, and discarding the supernatant; adding 500ul buffer to resuspend cells;
(2) Negative cells were magnetically sorted.
The MAC medium-sized magnetic head is tightly attached to the middle position of the magnetic frame, and an LS sorting column is placed; fully infiltrating the sorting column with 3ml buffer solution; dripping the marked marrow mononuclear cell suspension into a separation column from a middle position at a constant speed, avoiding generating bubbles, and collecting Lin - cells from the lower part of the separation column; washing the sorting column 3 times with 3ml buffer; counting cells of the collected Lin - suspension;
3. Lin -CD117+ cells were sorted by magnetic beads.
(1) Immunomagnetic beads labeled Lin - cells.
Centrifuging the Lin - cell suspension collected in the step 2, 300g for 5min, and discarding the supernatant; mu.l of magnetic bead sorting buffer was added per 10 8 cells; 200 μl CD117 microbead labeled Lin - cells were added; gently mixing and placing the mixture in a place for incubation for 15min at 4 ℃; adding 10ml buffer solution, 300 Xg, 10min, and discarding supernatant; add 500. Mu.l buffer for later use.
(2) Positive cells were magnetically sorted.
The MAC small-sized magnetic head is tightly attached to the middle position of the magnetic frame, and an MS sorting column is placed; fully infiltrating the sorting column with 500 μl buffer; dripping the Lin - cell suspension marked with the CD117 antibody into a separation column from the middle position at a constant speed to avoid generating bubbles; the column was washed 3 times with 500 μl buffer; removing the MS column combined with the positive cells from the magnetic field, and placing the MS column on a clean 15ml centrifuge tube; 1ml buffer solution is dripped into the center of the separation column at a constant speed, and positive cells with magnetic marks are pushed out by lightly pressurizing by a piston matched with an MS column; the collected Lin -CD117+ cells were subjected to cell counting.
3. Three-dimensional hydrogel cell culture system preparation, wherein the system components include: IMDM culture medium, fetal calf serum, sodium alginate, collagen I and growth factor; the content of each component is as follows: sodium alginate 0.25-0.50wt%, collagen I1-3mg/mL, IMDM culture medium solution 10mg/mL, fetal bovine serum 10v/v%, SCF 50ng/mL, flt3L 20ng/mL, TPO 20ng/mL.
1. Preparing IMDM culture medium containing 10v/v% of fetal bovine serum, 50ng/ml of SCF, 20ng/ml of Flt3L and 20ng/ml of TPO, and re-suspending hematopoietic stem cells by using the freshly prepared culture medium;
2. Precooling type I collagen acidic solution, 1N sodium hydroxide solution, 10xPBS, sodium alginate solution and 10-60mg/ml CaCl 2 solution, taking out, and placing on ice for operation;
3. taking 1-3mg of collagen, firstly adding 100 mu l of sodium alginate solution, quickly and uniformly mixing, and placing on ice for precooling for 2min;
4. Precooling, taking out, and adjusting pH to neutrality with sodium hydroxide solution;
5. adding 100 μl of 10xPBS, rapidly mixing, and pre-cooling;
6. Adding 5x10 5 freshly extracted hematopoietic stem cells, and gently mixing;
7. adding sterile water to make up the volume of the system to 1ml;
8. Spreading the collagen-cell mixed solution obtained in the step 2 in a 12-hole plate, wherein 1 ml/hole is placed in a cell incubator at 37 ℃ for 30min;
9. Taking out after gel formation, uniformly dripping 1ml 56mg/ml CaCl 2 onto the surface of the hydrogel, and placing the hydrogel in a cell incubator at 37 ℃ for crosslinking for 20min;
10. The well plate was removed, caCl 2 was aspirated, washed 3 times with hepes buffer, IMDM medium containing cytokines (SCF+TPO+Flt3L) was added, and the cells were cultured in a 37℃cell incubator.
4. Degradation of collagen hydrogel systems and cell separation.
1. Preparing collagenase solution.
Weighing 50-100mg collagenase powder, adding sterile deionized water to constant volume to 50ml, and preparing 1-2mg/ml collagenase solution; after the powder is completely dissolved, filtering and sterilizing by a disposable filter; collecting with sterile centrifuge tube, packaging, and storing at-20deg.C;
2. preparing 0.05-0.1mol/L sodium citrate buffer solution.
Weighing 4.17-8.34g of sodium citrate powder, and adding deionized water to a volume of 100ml; sterilizing at 120deg.C for 20min under high temperature and high pressure;
3. digestion of hydrogels.
Taking out a 12-well plate for hydrogel culture, discarding the culture solution of the supernatant, adding 1ml of sodium citrate buffer solution into each well, and incubating for 20min at 37 ℃; sucking the supernatant of the hydrogel, adding 1ml collagenase solution into each hole, and incubating for 1h at 37 ℃; after the collagen hydrogel was completely digested and dissolved, the cells were collected by centrifugation at 1000rpm for 5min with gentle shaking.
The three-dimensional hydrogel culture systems respectively constructed according to the three examples were subjected to elastic model characterization, and the results are shown in table 1:
TABLE 1
| Example 1 | Example two | Example III |
| 40.76±0.30(Pa) | 450.1±12.08(Pa) | 23.30±0.26(kPa) |
Referring to fig. 2A and 2B, according to the three-dimensional hydrogel culture system constructed by the scheme of the first embodiment, the second embodiment and the third embodiment, respectively, the cell compatibility test performed by the flow cytometry shows that: the cell survival rate is more than 60 percent, namely the cell has good cell compatibility. Specifically, the cell viability of example one was 66.0% (Q4 region), the cell viability of example three was 70.9% (Q4 region), and the cell viability of example three was 69.3% (Q4 region).
Referring to fig. 3, the three-dimensional hydrogel culture system (3D) constructed according to the first embodiment maintains the stem property of hematopoietic stem cells in vitro and promotes cell proliferation, compared to two-dimensional level culture (2D) and conventional fluid culture (NC) (cell culture for 24h, 48h, 72 h) by detecting stem cell surface markers through flow cytometry.
Referring to fig. 4A, a three-dimensional hydrogel culture system (3D) constructed in accordance with example one can regulate differentiation of macrophage lineage by bioinformatic analysis. Wherein, fig. 4A shows that three-dimensional hydrogel (3D) changes the differentiation direction of macrophage lineage compared to two-dimensional horizontal culture (2D), resulting in a cluster of more active macrophage subpopulations, i.e. "three-dimensional-macrophages"; FIG. 4B shows that "three-dimensional-macrophages" highly express a variety of chemokines and transcription factors.
The above-described embodiments by the present application have the following effects:
1. The invention develops a novel three-dimensional bone marrow niche bionic hydrogel by taking collagen I and sodium alginate as main materials. CaCl 2 with different concentrations is used as a cross-linking agent to realize the regulation and control of the rigidity of a system matrix, the culture environment with specific rigidity can be prepared according to different cell culture requirements, cells can be in a complete wrapping state, the mechanical strength required by cell culture is met, meanwhile, the cell-based composite material has the characteristics of good cell compatibility and easy degradation, and the in-vivo structure of the cells can be better simulated in vitro;
2. the hydrogel culture system prepared by the invention greatly shortens the preparation time, is convenient to operate, has low cost, does not need extra special equipment, and is convenient for expanding production;
3. The hydrogel culture system developed by the invention can maintain the proliferation and dryness of hematopoietic stem cells in vitro; and can be used for realizing the in vitro regulation and control of hematopoietic stem cells-monocytes-macrophages, and can generate specialized three-dimensional macrophages without adding induction factors, thereby having wide application prospects of tissue engineering.
The above description is only an example of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (3)
1. An application of a hematopoietic stem cell in-vitro 3D hydrogel culture system in promoting differentiation of hematopoietic stem cells into macrophages, wherein the 3D hydrogel culture system comprises the following components: type I collagen 1-3mg/mL, IMDM culture medium 10mg/mL, fetal bovine serum 10v/v%, SCF 50ng/mL, flt3L 20ng/mL, TPO 20ng/mL.
2. The use according to claim 1, wherein the method for constructing an in vitro 3D hydrogel culture system for hematopoietic stem cells comprises:
Obtaining primary marrow hematopoietic stem cells;
resuspending the hematopoietic stem cells with IMDM medium containing fetal bovine serum and cytokines;
Uniformly mixing an I-type collagen acidic solution, a 1N sodium hydroxide solution and 10 XPBS, adding hematopoietic stem cells after the PH is regulated to be neutral, and uniformly mixing to form hydrogel;
adding IMDM culture medium containing fetal bovine serum and cytokines into the hydrogel;
Preparing collagenase solution and sodium citrate buffer solution respectively, adding the sodium citrate buffer solution and the collagenase solution into the hydrogel respectively, dissolving the hydrogel and collecting cells so as to regulate and control the differentiation of macrophage lines;
The IMDM culture medium containing the fetal bovine serum and the cytokines is an IMDM culture medium containing 10v/v percent of the fetal bovine serum, 50ng/ml percent of SCF, 20ng/ml percent of Flt3L and 20ng/ml percent of TPO.
3. The use according to claim 2, wherein the step of obtaining primary bone marrow hematopoietic stem cells comprises:
collecting bone marrow, and collecting to obtain marrow mononuclear cells by a density gradient centrifugation method;
Magnetic beads separate Lin - progenitor cells from bone marrow mononuclear cells;
lin - CD117+ cells in bone marrow mononuclear cells were isolated by magnetic beads.
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