CN111187749A

CN111187749A - Artificial structure body for rapidly regenerating vascularized tissue, construction method and application

Info

Publication number: CN111187749A
Application number: CN201811352919.1A
Authority: CN
Inventors: 姚睿; 孙伟
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2018-11-14
Filing date: 2018-11-14
Publication date: 2020-05-22
Anticipated expiration: 2038-11-14
Also published as: CN111187749B

Abstract

The invention provides an artificial structure body for rapidly regenerating vascularized tissues and a construction method thereof. Wherein, the tissue-related cells are uniformly distributed in the cell/hydrogel microspheres, and the exterior of the microspheres contains a vascularized cell layer; the carrier material is uniformly distributed with cell/hydrogel microspheres, tissue-related cells, vascularized cells and growth factor sustained-release microspheres. The artificial structure can be cultured by a bioreactor to obtain a vascularized tissue precursor with a vascular-like network structure or transplanted into a body to regenerate vascularized functional tissues. The obtained tissue precursor or functional tissue has certain physiological functions and can be used for in vivo and in vitro researches such as tissue development, disease occurrence, drug development and detection, tissue regeneration and repair and the like.

Description

Artificial structure body for rapidly regenerating vascularized tissue, construction method and application

Technical Field

The invention relates to the technical field of biology, in particular to an artificial structure body for rapidly regenerating vascularized tissues and a construction method and application thereof.

Background

Tissue engineering began to be applied in the research field in the 80's of the 20 th century for the development and ultimate application of biosubstituents to restore, maintain or improve tissue function. Bioengineered tissues can be designed and prepared in the laboratory by tissue engineering. The engineered tissue may be implanted in vivo for the purpose of restoring, replacing, maintaining or enhancing tissue organ function.

The in vivo transplantation mode of the engineered tissue can be divided into two modes of traumatic transplantation and minimally invasive surgery. In clinical use, the engineered tissue must have a large volume on the order of centimeters and encapsulate a large number of cells to repair or replace the damaged tissue in vivo. High volume implants place high demands on the procedure, and still traumatic transplantation is the current common practice. The injection type minimally invasive surgery mode is popular with doctors and patients due to the characteristics of small trauma, low pain, low surgery complexity, hidden wound and the like.

Another major challenge for the survival of large volume implants in vivo is rapid and adequate revascularization, since when each cell is more than 200 μm away from the nearest blood vessel, there is a problem of insufficient supply of nutrients, eventually leading to cell necrosis. Without intervention of external conditions, the speed of the blood vessel growing into the implant is extremely slow, generally of the order of a few millimeters per day. The problem of vascularization is considered to be one of the bottleneck problems limiting the development of tissue engineering. The current successful tissue engineering products are mainly skin implants, which are characterized by large two-dimensional size, but small dimension in the height direction, and low requirement on vascularization.

There is a need for engineered tissues that can be used for minimally invasive injection grafting and rapid in vivo vascularization.

Disclosure of Invention

The invention aims to provide an artificial structure body for rapidly regenerating vascularized tissues, a construction method and application.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a method for constructing an artificial structure useful for rapid regeneration of vascularized tissue, comprising the steps of:

s1, preparing tissue cell hydrogel microspheres;

s2, culturing the tissue cell hydrogel microspheres in a cell culture solution to obtain tissue cell microspheres, wherein cells in the microspheres proliferate and/or differentiate into discrete cells or are in a cell cluster state;

s3, uniformly coating the coating material on the surface of the histiocyte microsphere prepared in the S2 to obtain the histiocyte microsphere with the coating;

s4, mixing the vascularized cells A with the tissue cell microspheres with the coatings prepared in S3, and culturing in a blood vessel cell culture solution for 3-5 days to obtain the tissue cell microspheres containing a blood vessel cell layer;

s5, preparing the slow release microspheres of the vascularization growth factor 1;

s6, preparation of artificial structure: and (3) mixing the histiocyte microspheres containing the vascular cell layer prepared in the step (S4), the vascularized growth factor 1 sustained-release microspheres prepared in the step (S5), the vascularized growth factor 2, the vascularized cells B, the histiocytes and a carrier material, and culturing in a mixed culture solution containing a vascular cell culture solution and a histiocyte culture solution to obtain the artificial structure body capable of quickly regenerating vascularized tissues.

Wherein the vascularized

growth factors

1 and 2 are the same or different vascularized growth factors; the vascularized cells A, B are the same or different vascularized cells.

In the present invention, the vascularized growth factors are any cytokines that promote capillary angiogenesis and promote vascular maturation. The

angiogenesis factors

1 and 2 are selected from at least one of vascular endothelial cell growth factor, basic fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, transforming growth factor, angiopoietin-1 and other blood vessel related growth factors, preferably vascular endothelial cell growth factor, basic fibroblast growth factor, epidermal growth factor and platelet-derived growth factor;

more preferably, the vascularized growth factor 1 is basic fibroblast growth factor and vascular endothelial growth factor, and the vascularized growth factor 2 is epidermal growth factor.

In some embodiments, the angiogenesis factor is encapsulated by a carrier material, a sustained release carrier, or the like, preferably by a sustained release carrier.

In the present invention, the vascularized cells A, B are selected from at least one of vascular endothelial cells, vascular endothelial progenitor cells, microvascular endothelial cells, vascular smooth muscle cells, vascular fibroblasts, mesenchymal stem cells, pericytes, etc., which may be obtained by tissue extraction or stem cell differentiation, and are preferably vascular endothelial cells or mesenchymal stem cells;

more preferably, the vascularized cells A, B are umbilical vein endothelial cells, mesenchymal stem cells, or endothelial progenitor cells.

In the present invention, the coating material is biodegradable and has bioactivity, cell adhesion and biocompatibility as natural biomaterials, including but not limited to collagen, fibrinogen and matrigel.

In the invention, the carrier material is a natural biological material and/or an artificial synthetic biological material with biocompatibility.

The natural biomaterial is at least one selected from gelatin, gelatin derivatives, alginate derivatives, cellulose-derived materials, agar, matrigel, collagen derivatives, amino acids, amino acid derivatives, proteoglycans, proteoglycan derivatives, glycoproteins and derived materials, hyaluronic acid derivatives, chitosan derivatives, DNA hydrogel materials, layer-connecting proteins, fibronectin, fibrin derivatives, silk fibroin derivatives, etc., preferably sodium alginate, gelatin or collagen.

The artificial biological material is at least one selected from polypropylene, polystyrene, polyacrylamide, polylactide, polyglycolide, polylactic acid-glycolic acid copolymer, polyhydroxy acid, polylactic acid-glycolic acid copolymer, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide and the like, and preferably is polylactic acid or lactic acid-glycolic acid copolymer.

The tissue cells are selected from at least one of tumor cells, fibroblasts, totipotent stem cells, pluripotent stem cells, multipotent stem cells, immune cells, chondrocytes, bone-derived cells, tenocytes, skeletal muscle cells, adipocytes, parenchymal hepatocytes, kupffer cells, stellate cells, bile duct epithelial cells, tumor cells, antral endothelial cells, pancreatic cells, cardiac muscle cells, nerve cells, skin cells, hair follicle cells, sweat gland cells and other cells of various tissue and organ origins, preferably nerve cells, pancreatic cells, cardiac muscle cells, skin cells or parenchymal hepatocytes.

In the method, the average diameter of the microspheres prepared by S1 is 50-1000 μm, the average diameter of the microspheres prepared by S5 is 10nm-100 μm, and the average diameter of the microspheres prepared by S5 is smaller than that of the microspheres prepared by S1. For example, the average diameter of the microspheres prepared in S1 may be 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm or any value therebetween. For example, the microspheres prepared in S4 have an average diameter of 10nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm or any value therebetween.

After the hydrogel microspheres are formed, uniformly mixing the coating material with the histiocyte hydrogel microspheres, and carrying out static culture or dynamic culture in a certain culture space until the coating material is fully combined with the surfaces of the microspheres. The culture space can be selected from various spaces formed by common culture tools in the field, such as centrifuge tubes, 12-well plates, and the like. The dynamic culture method can be selected from instruments commonly used in the field, such as a microgravity culture device, a perfusion culture device and bioreactor, a stirring culture device and bioreactor, a wave culture device and bioreactor, and the like.

The tissue cell mass having the coating layer prepared in step S3 is subjected to static culture or dynamic culture in a blood vessel cell culture solution, and vascularized cells are attached to the material coating layer to form a blood vessel cell layer. The culture space can be selected from various spaces formed by common culture tools in the field, such as centrifuge tubes, 12-well plates, and the like. The dynamic culture method can be selected from instruments commonly used in the field, such as a microgravity culture device, a perfusion culture device and bioreactor, a stirring culture device and bioreactor, a wave culture device and bioreactor, and the like. The culture medium and culture conditions employed are those commonly used in the art for culturing vascularized cells and/or tissue cells and are determined by one of skill in the art based on techniques well known in the art.

In the foregoing method, S5 preferably uses sodium alginate as the encapsulating material to prepare the microspheres. The final concentration of sodium alginate in the microspheres is 0.5% to 10%, for example 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, etc.

In the present invention, the microspheres can be produced by a pendant drop method, a non-adhesive self-assembly method, a high-voltage electrostatic spray method, or the like. S1 and S5 are preferably both prepared by non-contact high-voltage electrostatic method. The preparation of the microspheres by the non-contact high-voltage electrostatic method can be carried out by using a non-contact high-voltage electrostatic generator of CN 200910079726.8.

Through channels are naturally formed in the gaps between the tissue cell hydrogel microspheres. The formed through channel can be used as the starting point and the space guarantee of the regeneration of the blood vessel in the body and is quickly connected with the blood vessel in the body.

The construction method of different types of histiocyte microspheres comprises the following steps:

preparation of islet cells/hydrogel microspheres

Taking conventionally cultured human pancreatic cells, adopting a sodium alginate/collagen mixture as a carrier material, and preparing the pancreatic cells/hydrogel microspheres by using a high-voltage electrostatic spraying preparation device:

1. a solution of sodium alginate (Sigma-Aldrich, A0682) and a solution of collagen (Sigma, C7661) were mixed to final concentrations of 2% -6% (w/w) and 0.1-2mg/mL (w/v), respectively, of the carrier material solution.

2. Human pancreatic CELLs (Pricells, HUM-CELL-0058) were cultured at 1X 10⁵-5×10⁷Per mThe density of l is mixed with the carrier material solution evenly and then is filled into a disposable syringe with the volume of 1-20 ml.

3. Connecting a high-voltage electrostatic jet cell microsphere preparation device, wherein the high-voltage electrostatic generation device adopts an SA167-Y (Tianjin) high-voltage electric field generator and outputs 7-12 kV; clamping the syringe of step 2 in a propulsion unit: in a TS2-60 type injection pump manufactured by LongerPump, the distance between a needle tip and a copper sheet is 10-30mm, the propelling speed is 5-50ml/h, a needle with the inner diameter of 100-.

4. The cell microspheres were collected within 5min, washed twice with physiological saline and observed and recorded, and the cell microspheres were round and smooth in shape and had an average diameter of 300 μm.

5. The islet cell culture solution (DMEM medium (Gibco,11965) and DMEM/F-12 medium (Gibco,11320) was added with 5-50mM Nicotinamide (Sigma,72340), 0.5-10nM Activin A (R)&D,294-HG), 1-100nM Exendin-4(Sigma, E7144), 1-100nM Pentagastin (Sigma, B1636), 10-1000 pMhepetocyte growth factor (Sigma, SRP6014), 2% B-27supplement (Gibco,17504), 1% N-2supplement (Gibco, A13707), 1% streptomycin (Gibco,15140122)) at 37 deg.C, 5% CO₂And culturing the islet cells/hydrogel microspheres in a saturated humidity incubator for 1-20 days to obtain the sodium alginate/collagen hydrogel microspheres containing high-density islet cells.

The collagen material is uniformly coated on the surface of the microsphere by adopting the following steps.

6. And (3) uniformly mixing the collagen solution (Sigma, C7661, stock solution) and the cell microspheres obtained in the step (5) in a ratio of 5:1-1:5, and then filling the mixture into a centrifugal tube, wherein the cell microspheres are deposited at the bottom due to higher density. Centrifuging at the rotating speed of 600-1500r/min for 2-10min to ensure that the collagen solution is fully contacted with the islet cell microspheres at the bottom. And (3) performing static culture in a centrifugal tube for 12-72 hours, so that the collagen solution is uniformly distributed on the surfaces of the microspheres, the local microspheres are connected into a whole under the self-assembly action, and a local through channel is formed as a space for forming a vascular network.

The following steps are adopted to ensure that the vascularized cells are uniformly applied on the surface of the collagen material.

7. Culturing human adipose derived mesenchymal stem cells (HMSC-ad) in stem cell culture solution (high-glucose DMEM medium containing 10% fetal bovine serum and double antibody) conventionally, and taking the stem cells of passage 2-10 generations, and adding 10⁶-10⁸Resuspending in culture medium at a density of 1/mL, mixing with the microspheres obtained in step 6 at a volume ratio of 1:1, and performing static culture in a cell culture box at 37 ℃ and 5% CO2 and saturated humidity for 1-10 days to form the engineered pancreatic tissue containing the vascularized growth factors.

Second, preparation of liver parenchymal cell/hydrogel microspheres

Taking conventionally cultured human parenchymal hepatocytes, adopting a sodium alginate/gelatin system as a carrier material, and preparing the hepatocytes/hydrogel microspheres by using a high-voltage electrostatic spraying cell microsphere preparation device:

1. a solution of sodium alginate (Sigma-Aldrich, A0682) and a solution of gelatin (Sigma-Aldrich, G1890) in PBS buffer or physiological saline as carrier material solutions with final concentrations of 1% -6% (w/w) and 2% -6% (w/w), respectively, were mixed.

2. Hepatic parenchymal CELLs (Pricells, HUM-CELL-0036) were cultured at 1X 10⁵-5×10⁷The density of each ml is evenly mixed with the carrier material solution and then is filled into a disposable syringe with the volume of 1-20 ml.

4. The cell microspheres were collected within 5min, washed twice with physiological saline and observed and recorded, and the cell microspheres were round and smooth in shape and had an average diameter of about 400 μm.

5. Culturing with parenchymal hepatic cellsLiquid (high-glucose DMEM, 10% fetal bovine serum, 0.5mmol/L ascorbic acid, 0.5-10. mu. mol/L dexamethasone, 12. mu. mol/L insulin, 1% streptomycin stock solution) at 37 deg.C in 5% CO₂Culturing the liver parenchymal cell microspheres in a saturated humidity culture box for 1-20 days to obtain a high-concentration liver parenchymal cell cluster coated by sodium alginate/gelatin.

6. The collagen solution (Sigma, C7661) and the liver parenchymal cell microspheres are uniformly mixed in a ratio of 5:1-1:5 and then are filled into a centrifuge tube, and the cell microspheres sink to the bottom due to higher density. Centrifuging at the rotating speed of 600-1500r/min for 2-10min to ensure that the collagen solution is fully contacted with the hepatic parenchymal cell microspheres at the bottom. And (3) performing static culture in a centrifuge tube for 24-72 hours to ensure that the collagen solution is uniformly distributed on the surfaces of the microspheres.

7. Endothelial cells (ATCC, HUVEC) were routinely cultured in endothelial cell culture medium (F12 medium containing 2% fetal bovine serum, 0.1mg/ml heparin sodium and 0.04mg/ml endothelial cell growth aid ECGS), and well-grown cells were collected and 10 cells were cultured⁶-10⁸The cells/mL were resuspended in endothelial cell culture.

8. Preparing an endothelial cell layer by adopting a microgravity bioreactor: mixing the resuspended endothelial cells with the microspheres obtained in step 6 at a volume ratio of 1:5-5:1, inoculating 5-100mL of the mixture from the cell interface into a microgravity bioreactor (Synthecon, RCCS)^TM) In the reactor, the bubbles in the reactor are evacuated and the exhaust port is closed. The bioreactor assembly was placed at 37 ℃ with 5% CO₂Culturing in a cell culture box with saturated humidity at a rotation speed of 10-100rpm (preferably the minimum speed for suspending the microspheres without sinking) for 1-10 days, changing the liquid half a day, and uniformly adhering and growing endothelial cells on the surfaces of the microspheres to obtain the parenchymal hepatic cells/hydrogel microspheres in a mature period.

Preparation of cervical cancer tumor cell/hydrogel microspheres

Preparing human cervical cancer tumor cells/hydrogel microspheres from conventionally cultured cervical cancer tumors, and adopting a sodium alginate/vitronectin system as a microsphere material:

1. mixing the sodium alginate solution and the vitronectin solution into solutions with final concentrations of 1-6% and 0.1-10mg/ml respectively to serve as microsphere material precursor solutions.

2. Cervical cancer tumor cells (ATCC, H1HeLa) were cultured at 1X 10⁵-5×10⁷The density of each ml is evenly mixed with the precursor solution of the carrier material and then is filled into a disposable syringe with the volume of 1-20 ml.

3. And (3) preparing the cervical cancer tumor cells/sodium alginate/vitronectin microspheres by referring to the step 3 in the second step.

4. The cell microspheres were collected within 5min, washed twice with physiological saline and observed and recorded, and the cell microspheres were round and smooth in shape and had an average diameter of 450 μm.

5. Tumor cell culture medium (high-glucose DMEM, 10% fetal calf serum, 0.1% penicillin-streptomycin) was used at 37 deg.C and 5% CO₂Culturing the microspheres in a saturated humidity incubator for 1-10 days to obtain the high-concentration tumor cell microspheres wrapped by the sodium alginate/vitronectin.

6. The coating material coating layer was prepared by referring to the steps in the two above.

7. Endothelial cells (ATCC, HUVEC) were routinely cultured in endothelial cell culture medium (F12 medium containing 2% fetal bovine serum, 0.1mg/ml heparin sodium and 0.04mg/ml endothelial cell growth aid ECGS), and human cerebral adventitia fibroblasts (ATCC, HBVAF) were routinely cultured in fibroblast culture medium (RPMI 1640 medium containing 10% fetal bovine serum without HEPES). Taking endothelial cells with good growth state and human cerebral vascular adventitia fibroblast, both of which are 10⁶-10⁸The cells/mL were resuspended in endothelial-fibroblast culture medium (endothelial cell culture medium and fibroblast culture medium mixed at a 1:1 volume ratio).

8. Preparing a vascularized cell layer by adopting a microgravity bioreactor: mixing the resuspended endothelial cells and fibroblasts with the microspheres obtained in step 6 at a volume ratio of 1:1, inoculating 5-100mL of the mixture from the cell interface into a microgravity bioreactor (Synthecon, RCCS)^TM) In the reactor, the bubbles in the reactor are evacuated and the exhaust port is closed. The bioreactor device is placed at 37℃,5％CO₂Culturing in a cell culture box with saturated humidity at a rotation speed of 10-100rpm (preferably the minimum speed for suspending the microspheres without sinking) for 1-10 days, changing the liquid half a day, and uniformly adhering and growing the vascularized cells on the surfaces of the microspheres to obtain the cancer cells/hydrogel microspheres in the mature period.

Preparation of skin cell/hydrogel microspheres

1. Uniformly mixing human skin fibroblasts (ATCC, HSF), human skin melanocytes (Sciencell, HM) and human epidermal keratinocytes (ATCC, Adult epidermal keratinocytes) with microsphere precursor solution collagen (Sigma, C7661), wherein the final concentration of the two cells is 10⁵-10⁸The final concentration of the precursor solution is 0.1-10 mg/mL.

2. Loading the mixture obtained in step 1 into an Ink Jet cell PRINTER (CELLlink, BIO X PRINTER, Ink-Jet Print Head), printing the collagen carrier material containing the cells in microdroplets onto a non-adherent plastic substrate, at 37 ℃ and 5% CO₂And inversely culturing for 2 hours in a cell culture box with saturated humidity to finish the preparation process of the microspheres.

3. The skin cell microspheres were collected, washed twice with physiological saline, and cultured in a skin cell culture medium (DMEM/F-12 medium containing 10% fetal bovine serum, 4mM glutamine, 100u/ml penicillin, 100u/ml streptomycin, 0.1-20. mu.g/ml hydrocortisone, 1-50. mu.g/ml transferrin, 1-50. mu.g/ml insulin, 1-100ng/ml epidermal growth factor) for 1-15 days with half a change of the medium per day.

4. And (4) coating an endothelial cell layer on the surface of the skin cell microsphere by referring to the steps in the three steps to obtain the skin cell/hydrogel microsphere in the mature period.

The construction method of the growth factor sustained-release microspheres comprises the following steps:

a high-voltage electrostatic device is adopted to wrap vascular endothelial cell growth factors (Abcam, AB9571) and basic fibroblast growth factors (Abcam, AB9596) in material microspheres to form a slow-release system. The method comprises the following specific steps:

1. sodium alginate was formulated with physiological saline as a 1-8% (w/w) solution.

2. Mixing alkaline fibroblast growth factor and vascular endothelial growth factor with sodium alginate solution at concentration of 1-100ng/mL and 1-100ng/mL, and loading into disposable syringe with volume of 1-20 mL.

3. Connecting a high-voltage electrostatic cell microsphere preparation device, wherein the working voltage is 10-17kV, the distance between a needle point and a copper sheet is 15-30mm, the propelling speed is 50-50ml/h, a needle head with the inner diameter of 30-300 mu m is adopted, a collector is a disposable plastic culture dish (plastic or glass culture dish with various sizes can be used), the diameter of the collector is 60mm, and the curing solution is 100-400mmol/L calcium chloride solution (or barium chloride solution).

4. Collecting the slow release microspheres of the angiogenesis factors within 5min, washing the microspheres twice by using normal saline, and observing and recording, wherein the slow release microspheres of the angiogenesis factors have round and smooth shapes and the average diameter of 30 mu m.

5. Care was taken to keep the temperature as low as possible throughout the process.

The construction method of the artificial structure body for rapidly regenerating the vascularized tissue is specifically as follows:

preparation of artificial structure of tumor tissue

1. Uniformly mixing a gelatin (Sigma-Aldrich, G1890) solution (solvent PBS buffer solution) with a collagen solution (Sigma, C7661) according to a volume ratio of 1:5-5:1, wherein the mass fraction of the gelatin is 3% -20%, so as to form a carrier material precursor solution.

2. Respectively mixing the tumor cells/hydrogel microspheres containing the vascular cell layer obtained in the third step, the obtained angiogenesis factor sustained-release microspheres, the epidermal growth factor (Abcam, ab9697), the umbilical vein endothelial cells (ATCC, HUVEC) and the cervical cancer cells (ATCC, H1HELA) in a volume ratio of 5:1-20:1, 1:5-5:1, 5-50 ng/ml and 10⁵-10⁷one/mL and 10⁵-10⁷Uniformly mixing the carrier material precursor solution obtained in the step 1 with the concentration of one/mL, transferring the mixture into a 96-well plate in a volume of 50-200 mL/well, and carrying out 5% CO treatment at 37 DEG C₂And standing in a cell culture box with saturated humidity for 0.5-2h to complete the crosslinking process of the carrier material.

3. The artificial structure obtained in step 2 is cultured by uniformly mixing a vascular cell culture solution and a tumor cell culture solution in a volume ratio of 1:1 as a culture solution (the vascular cell culture solution is F12 culture medium containing 2% fetal calf serum, 0.1mg/ml heparin sodium and 0.04mg/ml endothelial cell growth auxiliary ECGS, the tumor cell culture solution is high-sugar DMEM, 10% fetal calf serum and 0.1% penicillin-streptomycin), and the culture solution is changed every 2-3 days and cultured for 2-20 days. Forming the artificial structure of the tumor-like tissue.

Preparation of artificial structure of nervous tissue of class II

Preparing neural stem cells/hydrogel microspheres by using a high-voltage electrostatic device, taking conventionally cultured neural stem cells (ATCC, ACS-5006) and adopting a sodium alginate/matrigel system as a carrier material:

1. a solution of sodium alginate and a solution of matrigel collagen (BD sciences, 356234) were mixed homogeneously at a volume ratio of 3:1 at low temperature, wherein the concentration of alginic acid was 2% -8% (w/w).

2. Human neural stem cells (ATCC, ACS-5006) were cultured at 1X 10⁵-5×10⁷The density of each ml is evenly mixed with the solution obtained in the step 1 and then is filled into a disposable syringe with the volume of 1-20 ml.

4. The cell microspheres are collected within 5min, washed twice with normal saline and observed and recorded, and the cell microspheres are round and smooth in shape and have an average diameter of 380 mu m.

5. Adopts neural cell differentiation culture solution (high-sugar DMEM, 5% fetal bovine serum, 10-300mg/ml NaHCO)₃5-50mmol/L D-glucose, 25U/ml penicillin-streptomycin, 0.5mmol/L L-glutamine, 1-10. mu. mol/L cytarabine) at 37 ℃ with 5% CO₂Inducing and differentiating the neural stem cell microspheres in a saturated humidity incubator for 5-15 days to obtain high-concentration neural cells coated by sodium alginate/matrigelAnd (4) clustering the cells.

6. Coating material coating layer preparation was described with reference to the procedure in one above.

7. Adhering the vascularized cell layer by referring to the steps in the first step to obtain the neural stem cell/hydrogel microspheres.

8. Uniformly mixing a collagen solution (Sigma, C7661) and a laminin solution (Sigma, L6274) according to a volume ratio of 1:5-5:1 to form a carrier material precursor solution.

9. Respectively mixing the neural stem cell/hydrogel microspheres obtained in the step 7, the obtained angiogenesis factor sustained-release microspheres, the epidermal growth factor (Abcam, ab9697), the umbilical vein endothelial cells (ATCC, HUVEC) and the neural stem cells (ATCC, ACS-5006) in a volume ratio of 5:1-20:1, 1:5-5:1, 5-50 ng/ml and 10⁵-10⁷one/mL and 10⁵-10⁷Uniformly mixing the carrier material precursor solution obtained in the step 8 with the concentration of one molecule/mL, transferring the mixture into a 96-well plate at the volume of 50-200 mL/well, and carrying out 5% CO treatment at 37 DEG C₂And standing in a cell culture box with saturated humidity for 0.5-8h to complete the crosslinking process of the carrier material.

10. The vascular cell culture solution and the nerve cell culture solution are uniformly mixed according to the volume ratio of 1:1 to serve as culture solution (the vascular cell culture solution is F12 culture medium containing 2% fetal calf serum, 0.1mg/ml heparin sodium and 0.04mg/ml endothelial cell growth auxiliary ECGS, and the nerve cell culture solution is high-sugar DMEM, 5% fetal calf serum and 10-300mg/ml NaHCO₃5-50mmol/L D-glucose, 25U/ml penicillin-streptomycin, 0.5mmol/L L-glutamine, 1-10. mu. mol/L cytarabine) the artificial structure obtained in step 9 was cultured by changing the culture medium every 2-3 days for 2-20 days. Forming the artificial structure of the nerve-like tissue.

In a second aspect, the present invention provides an artificial structure constructed as described above. The artificial structure comprises cell/hydrogel microspheres, tissue-related cells, vascularized cells, growth factor sustained-release microspheres and carrier materials. Wherein, the tissue-related cells are uniformly distributed in the cell/hydrogel microspheres, and the exterior of the microspheres contains a vascularized cell layer; the carrier material is uniformly distributed with cell/hydrogel microspheres, tissue-related cells, vascularized cells and growth factor sustained-release microspheres. Wherein, the carrier material can wrap the growth factor as a first-level factor slow-release system; the factor slow release microsphere has smaller diameter and is used as a secondary factor slow release system. The artificial structure can be cultured by a bioreactor to obtain a vascularized tissue precursor with a vascular-like network structure or transplanted into a body to regenerate vascularized functional tissues. The obtained tissue precursor or functional tissue has certain physiological functions and can be used for in vivo and in vitro researches such as tissue development, disease occurrence, drug development and detection, tissue regeneration and repair and the like. The invention can be used in the aspects of tissue engineering, regenerative medicine, in-vitro physiological model/pathological model/pharmacological model construction, tissue/organ/human body chip, cell biology or drug research and the like.

In a third aspect, the invention provides the use of the artificial structure in the construction of vascularized tissue. The artificial structure can be cultured by a bioreactor to obtain a vascularized tissue precursor with a vascular-like network structure, or transplanted (including but not limited to injection transplantation) into a body to regenerate vascularized functional tissues. For some situations, such as open wounds, where injection of the graft is not required, the tissue can also be injected rapidly into the lesion without affecting the surgical protocol.

In a fourth aspect, the present invention provides a method for constructing a vascularized tissue, comprising culturing the artificial structure in a bioreactor containing a mixed culture solution comprising a vascular cell culture solution and a tissue cell culture solution for 1 to 30 days (preferably 8 to 12 days).

The object of the invention can be further achieved by the following technical measures.

The artificial structure for rapidly regenerating vascularized tissue of the present invention is an engineered tissue useful for minimally invasive implantation and for rapidly regenerating vascularized tissue in vivo, said engineered tissue comprising more than one structural unit, wherein said structural units comprise a carrier material; tissue cells and vascularized growth factors (or no growth factors, only tissue cells) are distributed in the carrier material; the carrier material is coated with a material coating layer (or not coated), a vascular cell layer is adhered to the outside of the material coating layer, and through channels are formed in gaps between the structural units. The formed through channel can be used as the starting point and the space guarantee of the regeneration of the blood vessel in the body and is quickly connected with the blood vessel in the body.

The engineered tissue of the invention is an injection-type engineered tissue. In some embodiments, the engineered tissues of the invention are transplanted into the body by injection administration and revascularization is rapidly achieved in the body. Preferably, the engineered tissue of the present invention is implanted into the body using a disposable syringe.

In some embodiments, the building blocks are spherical.

In some embodiments, the structural units are microscale structural units.

In some embodiments, the average diameter of the structural units is from 50 μm to 1000 μm. For example, the average diameter of the structural units may be 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm or any value therebetween.

In some embodiments, the carrier material is a natural biomaterial and/or a synthetic biomaterial that is biodegradable and biocompatible.

The engineered tissue can be prepared according to the following method: tissue cells are wrapped by natural biological materials and/or artificial synthetic biological materials to be manufactured into micron-sized spherical structural units, gaps among the structural units are used as through channels for forming blood vessels, and a material layer (or no material layer) and a blood vessel cell layer are coated outside the structural units so as to realize rapid in-vivo blood vessel regeneration, and the method comprises the following steps:

(1) tissue cells and vascularized growth factors (or factors can be absent, only tissue cells) are wrapped in a carrier material to form tissue cell microspheres, (or stem cells and/or vascularized growth factors are wrapped in the carrier material, then directional differentiation inducing factors are added into a culture medium, and the stem cells are induced and differentiated into the required tissue cells to form the tissue cell microspheres);

(2) uniformly coating a material coating layer (or not) formed by biomaterial on the outer layer of the histiocyte microsphere;

(3) and a vascular cell layer is adhered outside the material coating layer.

The tissue cells or vascularized growth factors can be encapsulated in the carrier material by a variety of methods known to those skilled in the art, such as pendant drop method, non-adherent self-assembly method, high-pressure electrostatic spray method, etc., preferably high-pressure electrostatic spray method, as follows: tissue cells are treated with 10⁶～10⁷Mixing the density of each/ml and 1-5000 ng/ml of the vascularization growth factor (or not), extruding the mixture by using an injection pump, and dripping the mixture into a curing liquid by using the pulling force of a high-voltage electrostatic field to prepare the tissue cell microspheres containing or not containing the vascularization growth factor.

In some embodiments, the sodium alginate-based hydrogel material may be at least one of sodium alginate, sodium alginate/collagen, sodium alginate/gelatin, sodium alginate/fibrinogen, and the like.

In some embodiments, the final concentration of the sodium alginate based microsphere material is 0.5% to 10%, such as 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, and the like.

In some embodiments, the solidifying liquid is a salt solution containing divalent cations, such as calcium chloride, barium chloride, and the like, preferably a calcium chloride solution.

In some embodiments, the calcium chloride solution is used at a concentration of 100mM to 500mM, e.g., 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, or 500mM, etc.

In another aspect, the invention relates to the use of an engineered tissue in the preparation of a medicament for rapid in vivo vascularization of tissue.

In another aspect, the present invention relates to the use of an engineered tissue prepared according to the method of the invention in the preparation of a medicament for rapid in vivo vascularization of tissue.

In another aspect, the invention relates to the use of engineered tissue in the preparation of implants for the construction of physiological/pathological/pharmacological models, pharmacological studies, the treatment of diseases and for restoring, replacing, maintaining or improving the function of tissue organs.

In another aspect, the invention relates to the use of the engineered tissue prepared according to the method of the invention in the preparation of implants for the construction of physiological/pathological/pharmacological models, pharmacological studies, the treatment of diseases and for restoring, replacing, maintaining or improving the function of tissue organs.

In another aspect, the present invention relates to a method for rapidly regenerating vascularized tissue in vivo by engineering tissue, which comprises implanting the engineered tissue of the present invention into the body by minimally invasive injection and rapidly regenerating vascularized tissue in vivo.

In some embodiments, the methods comprise implanting the engineered tissue of the invention into the body by using a disposable syringe and rapidly achieving revascularization in the body.

In another aspect, the invention relates to a method of regenerating tissue having a functional vascular network in vivo, comprising implanting the engineered tissue of the invention into the body by minimally invasive injection, thereby forming tissue having a functional vascular network in the body.

In some embodiments, the methods comprise using a disposable syringe to implant the engineered tissue of the invention into the body, thereby forming a tissue with a functional vascular network in the body.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the first one contains two-stage slow releasing system of growth factor. The average diameter of the growth factor slow release microspheres is between 10nm and 100 mu m, so that the slow release of a certain growth factor can be realized; the carrier material can be wrapped with another growth factor, and the release rate is greater than that of the sustained release microspheres, so that the graded delivery of various growth factors can be realized.

And (II) injectable minimally invasive transplantation. The basic structural unit of the invention is hydrogel microsphere, the average diameter is between 50 μm and 1000 μm, when the injectable material is adopted as the carrier material, the injection minimally invasive transplantation can be realized, the wound is small, the postoperative recovery is fast, and the invention is easy to be accepted by patients.

And (III) can realize rapid in vivo blood vessel regeneration. The naturally existing through channels between the basic structural units and the vascularized cells coated on the outer layer are used as the starting point and the space guarantee of the in vivo blood vessel regeneration, can be quickly connected with the in vivo blood vessel, and realize the functional blood vessel network regeneration in a short time. In addition, the artificial structure body also comprises a two-stage growth factor slow release system which can promote capillary angiogenesis and blood vessel maturation so as to further promote in-vivo blood vessel regeneration and ensure that the blood vessel regeneration speed can reach as high as about 8mm²On the order of a day.

And (IV) the in vivo regeneration of large-volume tissues can be realized. By controlling the number of basic structural units \ growth factor sustained-release microspheres, hundreds to tens of thousands of spherical structural units can be injected at one time, and a large number of functional blood vessels regenerated among structural unit gaps can ensure centimeter magnitude and clinically apply in-vivo survival, remodeling and regeneration of tissues with related sizes.

Drawings

FIG. 1 is a schematic view of the structure of the artificial structure of the present invention. Wherein, 1: cell/hydrogel microspheres; 2: a tissue-associated cell; 3: growth factor slow release microspheres; 4: a growth factor; 5: a carrier material; 6: vascularizing the cells.

FIG. 2 is a pictorial view of an artificial structure prepared in examples 1 and 4 of the present invention. Wherein, A: at the initial stage of cell/hydrogel microsphere formation, islet cells (shown by black arrows) are wrapped in microspheres of carrier material composed of sodium alginate and collagen, a collagen coating layer is arranged on the surface of the carrier material, and vascular endothelial cells (shown by white arrows) are adhered to the surface of the coating layer of the collagen raw material. B: the microstructure of the collagen coating layer was observed by scanning electron microscopy. C: during the mature period of the cell/hydrogel microsphere, parenchymal hepatic cells (shown by black arrows) are wrapped in a carrier material consisting of sodium alginate and gelatin, and mesenchymal stem cells (shown by white arrows) are uniformly adhered to the surface of the carrier material in a layered manner. D: the artificial structural body picture comprises cervical cancer cells/hydrogel microspheres (shown by black arrows), vascularized growth factor slow-release microspheres (shown by white arrows), vascularized cells and cancer cells. A scale: 200 μm.

FIG. 3 is a graph showing the HE staining effect of the regenerated vascularized tissue 28 days after the in vivo transplantation of the artificial structure in example 9 of the present invention. Wherein, A: HE staining of implant sections showed regenerated pancreatic tissue in vivo. B: HE staining of implant sections revealed in vivo regenerated neural tissue, red functional regenerated blood vessels containing large numbers of red blood cells, as indicated by the black triangular arrows. C: HE staining of the implant sections revealed in vivo regenerated cervical carcinoma tumor tissue, red functional regenerated blood vessels containing a large number of red blood cells, as indicated by the black triangular arrows. D: immunofluorescent staining shows the effect of regenerating liver when implanted into an object, and positive staining with Albumin (Albumin) is the functional liver cell that regenerates.

FIG. 4 is a graph showing the result of quantitative determination of regenerated vascularized tissue 28 days after the in vivo transplantation of the artificial structure in example 9 of the present invention. Wherein, A: the invention relates to the identification of the density of regenerated blood vessels in an artificial structure body and a control group body. B: the invention relates to the identification of regenerated vascularized tissue in artificial structure and control group. Represents p <0.01, represents p < 0.001.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The terms:

as used herein, "engineered tissue transplantation" refers to a technique for repairing, improving or reconstructing the structure and/or function of a tissue or organ of a patient by transplanting a tissue containing living cells prepared by a tissue engineering technique. "engineered tissue" refers to tissue containing viable cells that has been prepared by engineering techniques. The active cells are preferably human cells, more preferably autologous cells.

The terms "vascularization" and "formation of a functional vascular network" are used interchangeably herein and refer to the process of forming blood vessels and capillaries in body tissue.

As used herein, "angiogenesis" is meant to include both mechanisms of angiogenesis and vasculogenesis. The former form new blood vessels by activating precursor cells of vascularized cells, particularly vascular endothelial cells. The latter is angiogenesis, which is a process in which the vascular cells of existing capillaries proliferate to form a neovascular network. The "angiogenic growth factor" of the present invention plays an important role in both of these mechanisms.

The terms "basic unit", "basic building block" and "building block" are used interchangeably herein and are the basic building blocks of the engineered tissues of the invention, the structure of which is shown in FIG. 1, and the engineered tissues of the invention contain more than one of the units.

As used herein, "tissue cells" refers to morphologically functional similar cells that form the basic structure or organ of the human or animal body, excluding vascularized cells. As used herein, "vascularized cells" refers to cells that form blood vessels.

The invention uses the vascularized growth factor slow-release microsphere which is a sphere formed by only wrapping the vascularized growth factor in a carrier material.

As used herein, "uniform" means that the properties, thickness, density, etc. of a material or substance are consistent or nearly consistent.

The 'hydrogel' used in the invention has a three-dimensional network structure, can absorb a large amount of water in water to swell, and continuously keeps the original structure without being dissolved after swelling. The hydrogel is similar to a living tissue material, has weak capacity of adhering protein and cells on the surface, and shows good biocompatibility when being contacted with blood, body fluid and human tissues. It is similar to extracellular matrix part in property, and can reduce friction and mechanical action on surrounding tissues after absorbing water, thereby obviously improving the biological function of the material.

Terms such as "comprising," "including," "containing," and "including" are not intended to be limiting. Furthermore, unless otherwise indicated, the absence of a numerical modification includes the plural, and "or", "or" means "and/or". Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The pulse bioreactor used in the examples below can be found in CN 200910079726.8.

EXAMPLE 1 preparation of islet cells/hydrogel microspheres

1. a solution of sodium alginate (Sigma-Aldrich, A0682) and a solution of collagen (Sigma, C7661) were mixed to final concentrations of 2% (w/w) and 1mg/mL (w/v), respectively, of the carrier material solution.

2. Human pancreatic CELLs (Pricells, HUM-CELL-0058) were treated with 10⁶The mixture with the density of each ml and the carrier material solution is evenly mixed and then is filled into a disposable syringe with the volume of 5 ml.

3. Connecting a high-voltage electrostatic spraying cell microsphere preparation device, wherein the high-voltage electrostatic generation device adopts an SA167-Y (Tianjin) high-voltage electric field generator and outputs the voltage of 10 kV; clamping the syringe of step 2 in a propulsion unit: in the model TS2-60 syringe pump manufactured by LongerPump, the distance between the needle tip and the copper sheet is 10mm, the propelling speed is 20ml/h, a needle head with the inner diameter of 191 mu m is adopted, the collector is a disposable plastic culture dish with the diameter of 60mm (plastic and glass culture dishes with various sizes can be used), and the solidifying solution is 200mmol/L calcium chloride solution (or barium chloride solution).

5. 10mM Nicotinamide (Sigma,72340), 2nM Activin A (R) was added to islet cell culture (DMEM medium (Gibco,11965) and DMEM/F-12 medium (Gibco,11320)&D,294-HG), 10nMexendin-4(Sigma, E7144), 10nM Pentagastatrin (Sigma, B1636), 100pM hepatocyte growth factor (Sigma, SRP6014), 2% B-27supplement (Gibco,17504), 1% N-2supplement (Gibco, A13707), 1% streptomycin(Gibco,15140122)) 5% CO at 37 deg.C₂And culturing the islet cells/hydrogel microspheres in a saturated humidity incubator for 5 days to obtain the sodium alginate/collagen hydrogel microspheres containing high-density islet cells.

6. And (3) uniformly mixing the collagen solution (Sigma, C7661, stock solution) and the cell microspheres obtained in the step (5) in a ratio of 1:1, and then filling the mixture into a centrifuge tube, wherein the cell microspheres are deposited at the bottom due to higher density. Centrifuging at the rotating speed of 1000r/min for 4min to ensure that the collagen solution is fully contacted with the islet cell microspheres at the bottom. And (3) standing and culturing for 24 hours in a centrifuge tube, so that the collagen solution is uniformly distributed on the surfaces of the microspheres, the local microspheres are connected into a whole through the self-assembly effect, and a local through channel is formed as a space for forming a vascular network.

7. Culturing human adipose-derived mesenchymal stem cells (HMSC-ad) in stem cell culture solution (high-glucose DMEM medium containing 10% fetal bovine serum and double antibody) conventionally, and taking the stem cells of passage 3, and adding 10⁷Resuspending in culture medium at a density of 1/mL, mixing with the microspheres obtained in step 6 at a volume ratio of 1:1, and mixing at 37 deg.C with 5% CO₂And statically culturing in a cell culture box with saturated humidity for 3 days to form the engineered pancreatic tissue containing the vascularized growth factors. After 1 day of static culture, observation was performed under an optical microscope, as shown in FIG. 2A.

EXAMPLE 2 preparation of hepatocyte/hydrogel microspheres

1. a solution of sodium alginate (Sigma-Aldrich, A0682) and a solution of gelatin (Sigma-Aldrich, G1890) in PBS buffer or physiological saline as carrier material solutions at final concentrations of 2% (w/w) and 3% (w/w), respectively, were mixed.

2. Hepatic parenchymal CELLs (Pricells, HUM-CELL-0036) were treated with 10⁶Density of one/mlThe mixture was uniformly mixed with the carrier material solution and filled into a disposable syringe having a volume of 10 ml.

5. Adopting hepatocyte culture medium (high-glucose DMEM, 10% fetal calf serum, 0.5mmol/L ascorbic acid, 1 μmol/L dexamethasone, 12 μmol/L insulin, 1% streptomycin stock solution) at 37 deg.C with 5% CO₂Culturing the liver parenchymal cell microspheres in a saturated humidity incubator for 1 day to obtain a high-concentration liver parenchymal cell cluster coated by sodium alginate/gelatin.

6. Collagen solution (Sigma, C7661) and liver parenchymal cell microspheres are mixed uniformly in a ratio of 1:1 and then are filled into a centrifuge tube, and the cell microspheres sink to the bottom due to higher density. Centrifuging at 1000r/min for 4min to make the collagen solution fully contact with the bottom hepatic parenchymal cell microspheres. And (5) standing and culturing for 48 hours in a centrifuge tube, so that the collagen solution is uniformly distributed on the surfaces of the microspheres.

And observing the collagen coating condition by adopting a scanning electron microscope. After the coated cell microspheres are fixed by 1mol/L sodium arsenate buffer solution containing 2% of glutaraldehyde, 3% of paraformaldehyde and 5% of sucrose, the morphology of the collagen layer on the surfaces of the microspheres is observed by using a FEI Quanta 200 scanning electron microscope (Netherlands). The results show that the surface of the microspheres was uniformly coated with a layer of collagen fibers, as shown in fig. 2B.

7. Endothelial cells (ATCC, HUVEC) were routinely cultured in endothelial cell culture medium (F12 medium containing 2% fetal bovine serum, 0.1mg/ml heparin sodium and 0.04mg/ml endothelial cell growth aid ECGS), and well-grown cells were collected and 10 cells were cultured⁷The cells/mL were resuspended in endothelial cell culture.

8. Preparing an endothelial cell layer by adopting a microgravity bioreactor: mixing the resuspended endothelial cells with the microspheres from step 6 at a volume ratio of 1:1, and inoculating 20mL of the mixture from the cell interface into a microgravity bioreactor (Synthecon, RCCS)^TM) In the reactor, the bubbles in the reactor are evacuated and the exhaust port is closed. The bioreactor assembly was placed at 37 ℃ with 5% CO₂Culturing in a cell culture box with saturated humidity at a rotating speed of 20rpm (preferably a minimum speed for suspending the microspheres without sinking) for 3 days, changing the liquid half a day, and uniformly adhering and growing endothelial cells on the surfaces of the microspheres to obtain the liver parenchymal cells/hydrogel microspheres in a mature period. As shown in fig. 2C.

EXAMPLE 3 preparation of cervical cancer tumor cells/hydrogel microspheres

1. the sodium alginate solution and the vitronectin solution were mixed to final concentrations of 2% and 1mg/ml, respectively, as microsphere material precursor solutions.

2. Cervical cancer tumor cells (ATCC, H1HeLa) were treated with 10⁷The mixture was uniformly mixed with the carrier material precursor solution at a density of one piece/ml and then filled into a disposable syringe having a volume of 10 ml.

3. Cervical cancer tumor cells/sodium alginate/vitronectin microspheres were prepared according to step 3 of example 2.

5. Tumor cell culture medium (high-glucose DMEM, 10% fetal calf serum, 0.1% penicillin-streptomycin) was used at 37 deg.C and 5% CO₂Culturing the microspheres in a saturated humidity incubator for 3 days to obtain alginic acidSodium/vitronectin encapsulated high concentration tumor cell microspheres.

6. The coating material coating layer was prepared by referring to the procedure in example 2.

7. Endothelial cells (ATCC, HUVEC) were routinely cultured in endothelial cell culture medium (F12 medium containing 2% fetal bovine serum, 0.1mg/ml heparin sodium and 0.04mg/ml endothelial cell growth aid ECGS), and human cerebral adventitia fibroblasts (ATCC, HBVAF) were routinely cultured in fibroblast culture medium (RPMI 1640 medium containing 10% fetal bovine serum without HEPES). Taking endothelial cells with good growth state and human cerebral vascular adventitia fibroblast, both of which are 10⁶The cells/mL were resuspended in endothelial-fibroblast culture medium (endothelial cell culture medium and fibroblast culture medium mixed at a 1:1 volume ratio).

8. Preparing a vascularized cell layer by adopting a microgravity bioreactor: mixing the resuspended endothelial cells and fibroblasts at a volume ratio of 1:1 with the microspheres obtained in step 6, and inoculating 20mL of the mixture from the cell interface into a microgravity bioreactor (Synthecon, RCCS)^TM) In the reactor, the bubbles in the reactor are evacuated and the exhaust port is closed. The bioreactor assembly was placed at 37 ℃ with 5% CO₂Culturing in a cell culture box with saturated humidity at a rotating speed of 20rpm (preferably the minimum speed for suspending the microspheres without sinking) for 3 days, changing the liquid half a day, and uniformly adhering and growing the vascularized cells on the surfaces of the microspheres to obtain the cancer cells/hydrogel microspheres in a mature period.

Example 4 preparation of skin cell/hydrogel microspheres

1. Uniformly mixing human skin fibroblasts (ATCC, HSF), human skin melanocytes (Sciencell, HM) and human epidermal keratinocytes (ATCC, Adult epidermal keratinocytes) with microsphere precursor solution collagen (Sigma, C7661), wherein the final concentration of the two cells is 10⁶one/mL, the final concentration of the precursor solution was 1 mg/mL.

3. The skin cell microspheres were collected, washed twice with physiological saline, and cultured in a skin cell culture medium (DMEM/F-12 medium containing 10% fetal bovine serum, 4mM glutamine, 100u/ml penicillin, 100u/ml streptomycin, 0.4. mu.g/ml hydrocortisone, 5. mu.g/ml transferrin, 5. mu.g/ml insulin, 10ng/ml epidermal growth factor) for 3 days with half a change per day.

4. The endothelial cell layer was coated on the surface of the skin cell microspheres according to the procedure of example 3 to obtain mature-stage skin cell/hydrogel microspheres.

EXAMPLE 5 preparation of growth factor sustained-Release microspheres

1. sodium alginate was formulated as a 1.5% (w/w) solution in physiological saline.

2. The basic fibroblast growth factor and the vascular endothelial growth factor are uniformly mixed with the sodium alginate solution at the concentration of 2ng/mL and 10ng/mL and then filled into a disposable syringe with the volume of 10 mL.

3. Connecting a high-voltage electrostatic cell microsphere preparation device, wherein the working voltage is 12kV, the distance between a needle point and a copper sheet is 20mm, the propelling speed is 30ml/h, a needle head with the inner diameter of 191 mu m is adopted, a collector is a disposable plastic culture dish (plastic and glass culture dishes with various sizes can be used) with the diameter of 60mm, and a curing solution is 200mmol/L calcium chloride solution (or barium chloride solution).

EXAMPLE 6 preparation of Artificial Structure of tumor tissue of type

1. Gelatin (Sigma-Aldrich, G1890) solution (solvent PBS buffer) with the mass fraction of 10% and collagen solution (Sigma, C7661) were uniformly mixed in a volume ratio of 1:1 to form a carrier material precursor solution.

2. The tumor cells/hydrogel microspheres containing vascular cell layer obtained in example 3, the sustained-release microspheres of the angiogenic growth factor obtained in example 5, the epidermal growth factor (Abcam, ab9697), the umbilical vein endothelial cells (ATCC, HUVEC) and the cervical cancer cells (ATCC, H1HELA) were added in the volume ratio of 10:1, 2:1, 10ng/ml, 10⁶one/mL and 10⁶After the mixture of the carrier material precursor solution obtained in step 1 and the carrier material precursor solution was uniformly mixed, the mixture was transferred into a 96-well plate at a volume of 100 mL/well and then subjected to a reaction at 37 ℃ with 5% CO₂And standing for 1h in a cell culture box with saturated humidity to finish the cross-linking process of the carrier material.

3. The artificial structure obtained in step 2 was cultured in a medium (vascular cell medium: F12 medium containing 2% fetal bovine serum, 0.1mg/ml heparin sodium and 0.04mg/ml endothelial cell growth assistant ECGS; tumor cell medium: high-sugar DMEM, 10% fetal bovine serum, 0.1% penicillin-streptomycin) prepared by mixing vascular cell medium and tumor cell medium at a volume ratio of 1:1, and the medium was changed every 2-3 days for 10 days.

EXAMPLE 7 preparation of Artificial Structure of neural tissue

1. a solution of sodium alginate and a solution of matrigel collagen (BD sciences, 356234) were mixed homogeneously at a volume ratio of 3:1 at low temperature, wherein the concentration of alginic acid was 3% (w/w).

2. Mixing human neural stem cells (ATCC, ACS-5006) at a ratio of 10⁶The mixture with the density of each ml and the carrier material solution is evenly mixed and then is filled into a disposable syringe with the volume of 5 ml.

5. Neural cell differentiation medium (high-sugar DMEM, 5% fetal bovine serum, 100mg/ml NaHCO)₃20mmol/L D-glucose, 25U/ml penicillin-streptomycin, 0.5mmol/L L-glutamine, 2. mu. mol/L cytarabine) at 37 ℃ with 5% CO₂Inducing and differentiating the neural stem cell microspheres in a saturated humidity incubator for 8 days to obtain a high-concentration neural cell cluster wrapped by sodium alginate/matrigel.

6. The coating material coating layer was prepared according to the procedure described in example 1.

7. The neural stem cells/hydrogel microspheres were obtained by adhering a vascularized cell layer according to the procedure described in example 1.

8. The collagen solution (Sigma, C7661) and the laminin solution (Sigma, L6274) were mixed uniformly in a volume ratio of 1:1 to form a support material precursor solution.

9. Respectively taking the neural stem cell/hydrogel microspheres obtained in the step 7, the vascularized growth factor sustained-release microspheres obtained in the example 5, epidermal growth factor (Abcam, ab9697), umbilical vein endothelial cells (ATCC, HUVEC) and neural stem cells (ATCC, ACS-5006) in volume ratios of 10:1, 2:1, 10ng/ml and 10⁶one/mL and 10⁶After the mixture of the carrier material precursor solution obtained in step 8 and the carrier material precursor solution was uniformly mixed, the mixture was transferred into a 96-well plate at a volume of 100 mL/well and then subjected to a reaction at 37 ℃ with 5% CO₂And standing for 1h in a cell culture box with saturated humidity to finish the cross-linking process of the carrier material.

10. The vascular cell culture solution and the nerve cell culture solution are uniformly mixed in a volume ratio of 1:1 to serve as a culture solution (the vascular cell culture solution is F12 culture medium containing 2% fetal calf serum, 0.1mg/ml heparin sodium and 0.04mg/ml endothelial cell growth auxiliary ECGS; and the nerve cells are cultured in a culture mediumThe culture solution is as follows: high-sugar DMEM, 5% fetal bovine serum, 100mg/ml NaHCO₃20mmol/L D-glucose, 25U/ml penicillin-streptomycin, 0.5mmol/L L-glutamine, 2. mu. mol/L cytarabine) and culturing the artificial structure obtained in step 9 for 10 days with changing the culture medium every 2 to 3 days. Forming the artificial structure of the nerve-like tissue.

Example 8 bioreactor culture Artificial Structure

The pulse bioreactor is referred to CN 200910079726.8.

A peristaltic pump JD-200 produced in the service department of stainless steel fittings of Chongyang Zhongcheng in Chongzhou city is adopted to provide corresponding circulating power, the working voltage of the peristaltic pump JD-200 is set to be 12V, and the flow rate of the peristaltic pump JD-200 is set to be 60 ml/min; the direct current motor is a motor ZGB37RH52i produced by Beijing Aix company, and the working voltage is set to be 12V, and the rotating speed is set to be 100 r/min; a 100ml syringe is adopted; the self-made guide rod, the slide block guide rail and all the components, such as the direct current motor, the guide rod, the slide block guide rail and the injector, are fixed on the bottom plate by the self-made bracket to connect all the components.

The culture solution circulating part consists of a culture solution bottle, a peristaltic pump and a culture box, all parts are connected by a silicone tube, and the culture solution is pumped from the culture solution bottle to the culture box (built-in engineering tissue) by the peristaltic pump through the silicone tube and then flows back to the culture solution bottle through the silicone tube; the guide rail sliding block, the injector and the direct current motor form a pulsating part, the direct current motor is connected with the guide rail sliding block to push the injector piston to reciprocate, and the injector is connected with the liquid outlet end of the peristaltic pump and then connected with the culture box, so that pulsating flow is formed; the pressure gauge is arranged on the culture box and detects the culture fluid pressure in the tissue arranged in the culture box.

Before in vitro culture, connecting pipes and injectors of the pulse bioreactor are disassembled, and high-temperature and high-pressure sterilization is utilized. Then the pulse bioreactor is connected, the peristaltic pump is connected with the direct current motor, firstly, a small amount of 75% alcohol is added into the culture solution bottle, and the culture solution bottle is sterilized by flowing alcohol in a pulse circulation system; the alcohol was decanted and a sterile volume of PBS solution was added to the flask and the solution was used to rinse the residual alcohol.

The power was turned off, and then the culture solution to be used for the culture was added to the culture solution bottle, and the artificial tumor-like tissue construct prepared in example 6 was clamped by sterilized forceps and connected to the joint of the culture box. To securely attach the engineered tissue to the connectors, both ends of the engineered tissue are secured with sterilized threads. After the pulse bioreactor system is completely connected, the power supply is switched on, the voltage of the peristaltic pump is adjusted to 12V, the pressure at the artificial tissue is adjusted to 0.1Mpa, and then the pulse bioreactor can be continuously operated to carry out pulse culture on the engineered tissue.

The voltage and the tissue pressure are kept during the culture process, so that the pulse frequency is controlled at 100 times/min during the linear control pulse culture process.

After the direct current motor operates stably, the slide block is driven to push the syringe piston to reciprocate on the guide rail, the culture solution is sucked from the culture solution bottle when the piston is pulled out, and the sucked culture solution is injected into a circulating system formed by the peristaltic pump to flow through the engineered tissue in the culture box and flow back to the culture solution bottle when the culture solution is extruded. The pressure at the site of culturing the engineered tissue can be adjusted by adjusting the amount of culture solution aspirated and extruded each time by the syringe. Therefore, the peristaltic pump and the direct current motor continuously move, and the pulse bioreactor provides a pulse circulation culture fluid flow to realize the pulse culture of the engineering tissue.

The artificial structure is cultured in a pulsating mode for 1-30 days (preferably 5 days), and the liquid is changed half a day to obtain the pancreas-like, liver-like, nerve-like, tumor-like and skin-like artificial structures.

Morphology of the artificial structures was observed with CK40 inverted light microscope (japan). Under the microscope, the tumor cells/hydrogel microspheres, growth factor sustained-release microspheres, vascularized cells and tumor cells are uniformly distributed in the carrier material, as shown in fig. 2D. The regeneration speed of blood vessels can reach about 8mm²On the order of a day.

EXAMPLE 9 Effect of the artificial Structure after transplantation

The pancreas-like, liver-like, tumor-like and neuroid artificial structures prepared in example 8 were implanted subcutaneously into immunodeficient nude mice (BALB/c-nude, N ═ 4, vildagliptin). Control group materials and cell types, concentrations and amountsConsistent with the experimental group, but lack the cell microsphere structure and the growth factor sustained release microsphere structure. At 28 days post implantation, injection site samples were picked, paraffin sections were sectioned, immunohistochemical he (sigma) stained, and sections of the implants were viewed under an optical microscope (DP70, Olympus), as shown in fig. 3A-D. As can be seen from the results of in vivo transplantation, the engineered tissue provided by the invention obviously and rapidly regenerates a plurality of tissues in vivo, the tissues have morphological characteristics and protein expression of corresponding tissues, and the existence of a large number of red blood cells is observed, so that abundant functional blood vessels are regenerated. Each sample was taken 3 random fields and the density of vessels generated in the structure was obtained by quantitative analysis according to HE staining pictures, as shown in fig. 4A. 3 random fields were selected for each sample, and the proportion of neogenetic tissue generated in the structure occupying the fields was obtained by quantitative analysis according to HE staining pictures, as shown in fig. 4B. According to the detection result, the artificial structure provided by the invention obviously regenerates vascularized tissues in vivo, and the density and proportion of the new blood vessels are obviously different from those of a control group. The regeneration speed of blood vessels can reach about 8mm²On the order of a day.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for constructing an artificial structure for rapid regeneration of vascularized tissue, comprising the steps of:

s1, preparing tissue cell hydrogel microspheres;

s6, preparation of artificial structure: mixing the histiocyte microspheres containing the vascular cell layer prepared in S4, the vascularized growth factor 1 sustained-release microspheres prepared in S5, the vascularized growth factor 2, the vascularized cells B, the histiocytes and a carrier material, and culturing in a mixed culture solution containing a vascular cell culture solution and a histiocyte culture solution to obtain the artificial structure body capable of rapidly regenerating vascularized tissues;

wherein the vascularized growth factors 1 and 2 are the same or different vascularized growth factors; the vascularized cells A, B are the same or different vascularized cells;

the coating material is biodegradable and is a natural biological material with bioactivity, cell adhesion and biocompatibility;

the carrier material is a natural biological material and/or an artificial synthetic biological material with biocompatibility.

2. The method of claim 1, wherein the microspheres prepared in S1 have an average diameter of 50-1000 μm, the microspheres prepared in S5 have an average diameter of 10nm-100 μm, and the microspheres prepared in S5 have an average diameter smaller than that of S1.

3. The method of claim 1, wherein the angiogenic growth factors 1, 2 are selected from at least one of vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, transforming growth factor, angiopoietin-1, preferably vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, platelet-derived growth factor;

4. The method of claim 1, wherein the vascularized cells A, B are selected from at least one of vascular endothelial cells, vascular endothelial progenitor cells, microvascular endothelial cells, vascular smooth muscle cells, vascular fibroblasts, mesenchymal stem cells, pericytes, preferably from vascular endothelial cells, mesenchymal stem cells;

more preferably, the vascularized cells A, B are umbilical vein endothelial cells, mesenchymal stem cells, or endothelial progenitor cells; and/or

The tissue cell is selected from at least one of tumor cell, fibroblast, pluripotent stem cell, multipotent stem cell, immune cell, chondrocyte, bone-derived cell, tendon cell, skeletal muscle cell, fat cell, liver parenchyma cell, kupffer cell, stellate cell, bile duct epithelial cell, tumor cell, liver sinus endothelial cell, pancreatic cell, cardiac muscle cell, nerve cell, skin cell, hair follicle cell and sweat gland cell, preferably nerve cell, pancreatic cell, cardiac muscle cell, skin cell or liver parenchyma cell.

5. The construction method according to claim 1, wherein the coating material is selected from at least one of collagen, fibrinogen, matrigel; and/or

The natural biomaterial is selected from at least one of gelatin, gelatin derivatives, alginate derivatives, cellulose-derived materials, agar, matrigel, collagen derivatives, amino acids, amino acid derivatives, proteoglycans, proteoglycan derivatives, glycoproteins and derived materials, hyaluronic acid derivatives, chitosan derivatives, DNA hydrogel materials, layer-connecting proteins, fibronectin, fibrin derivatives, silk fibroin derivatives, preferably sodium alginate, gelatin or collagen; and/or

The artificial synthetic biomaterial is at least one selected from polypropylene, polystyrene, polyacrylamide, polylactide, polyglycolide, polylactic acid-glycolic acid copolymer, polyhydroxy acid, polylactic acid-glycolic acid copolymer, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate and polyethylene oxide, and preferably is polylactic acid or lactic acid-glycolic acid copolymer.

6. The method for constructing microspheres of claim 1, wherein S5 is prepared from sodium alginate as a capsule forming material.

7. The method of any one of claims 1-6, wherein both S1 and S5 are used to prepare microspheres by non-contact high-pressure electrostatic method.

8. An artificial structure constructed according to the method of any one of claims 1 to 7.

9. Use of the artificial structure according to claim 8 for the construction of vascularized tissues.

10. A method for constructing a vascularized tissue, characterized in that the artificial structure according to claim 8 is cultured in a bioreactor containing a mixed culture solution comprising a culture solution of vascular cells and a culture solution of tissue cells for 1 to 30 days.