CN107096070B

CN107096070B - Decellularized lung scaffold and preparation method thereof

Info

Publication number: CN107096070B
Application number: CN201710137190.5A
Authority: CN
Inventors: 黑飞龙; 马金辉; 鞠志海; 于洁; 乔叶薷; 候陈玮; 王晨; 陈书弘; 罗富良
Original assignee: Fuwai Hospital of CAMS and PUMC
Current assignee: Fuwai Hospital of CAMS and PUMC
Priority date: 2017-03-09
Filing date: 2017-03-09
Publication date: 2020-08-21
Anticipated expiration: 2037-03-09
Also published as: CN107096070A

Abstract

The invention provides a decellularized lung scaffold which is prepared by adopting sodium dodecyl ether sulfate and DNase I solution for perfusion treatment. The acellular lung scaffold prepared by the langendorff perfusion system is cleanly removed, the three-dimensional structure and extracellular matrix proteins are completely reserved, and the allogeneic subcutaneous transplantation has good fusibility, cellularization and vascularization after 6 weeks. The preparation method has high efficiency and simple steps, can carry out quantitative production, and has good application prospect in the field of tissue engineering lung and loose organs.

Description

Decellularized lung scaffold and preparation method thereof

Technical Field

The invention relates to the field of tissue engineering artificial lung, in particular to a decellularized lung scaffold and a preparation method thereof.

Background

At present, due to environmental factors, genetic factors, personal factors and the like, lung diseases such as Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF), alpha-1 antitrypsin deficiency, pulmonary arterial hypertension and the like are increasing, and for these patients, lung transplantation is the only treatment method with positive effect. But the development of lung transplantation is restricted due to severe donor deficiency, immune rejection, disease transmission, low long-term survival rate and the like. Currently, only 3700 worldwide lung transplants are in shortage every year, and the demand is far from being met. With the development of tissue engineering and regenerative medicine, especially the successful clinical application of tissue engineering skin, blood vessels, bladder, trachea and the like, the research of tissue engineering artificial lung is greatly encouraged, and a new direction is brought to lung transplantation.

The general process of constructing tissue engineering artificial lung includes the first obtaining ideal rack, the subsequent planting host cell, cell culture and final transplanting to host body to form one artificial lung with gas exchange function. Obtaining an ideal scaffold is critical for later cell seeding and in vivo transplantation.

Currently, sources for obtaining stents are: acellular scaffolds, natural scaffolds, synthetic scaffolds, 3D printing scaffolds, and the like. Among them, the decellularized scaffold has been widely used in clinical applications, and does not require any immunosuppressive agent, which is the most promising.

At present, the vibration method and the perfusion method are commonly used for preparing the acellular scaffold. The concussion method is too violent, seriously damages the three-dimensional structure of the tissue and is not suitable for the lung with loose structural tissue. The perfusion method is most suitable for preparing the acellular lung scaffold by perfusing a detergent and is widely applied to tissue engineering. The detergent commonly used at present is difficult to remove cells in the lung fully, and simultaneously, the three-dimensional structure of the lung is perfectly kept. Excessive cell retention after transplantation can lead to severe immune rejection. On the other hand, the internal components of the decellularized scaffold are extracellular matrix proteins, and the extracellular matrix proteins are lost too much, so that the natural structure and function of the scaffold are lost, and the cellularization and vascularization are influenced; the respiratory membrane is seriously broken, and the gas exchange capacity is difficult to maintain; transplanted into the body, the receptor has difficulty in long-term survival.

From the above, it can be seen that the search for an ideal detergent is a key point in the preparation of acellular lung scaffolds.

Disclosure of Invention

In order to overcome the technical problem, the invention provides a decellularized lung scaffold.

The invention also aims to provide a preparation method of the acellular lung scaffold.

In order to prepare an ideal acellular lung scaffold, the invention is realized by the following technical scheme: the novel detergent was infused using the langendorff infusion system: sodium Lauryl Ether Sulfate (SLES) was co-perfused with DNase i solution. The invention firstly utilizes the detergent SLES to prepare the lung stent, and simultaneously improves the application method of DNase I.

In order to achieve the above object, the acellular lung scaffold is prepared by using Sodium Lauryl Ether Sulfate (SLES) and DNase I solution for perfusion treatment.

Wherein the volume concentration of the Sodium Lauryl Ether Sulfate (SLES) is 0.1-1% (ml/ml).

The concentration of the DNase I solution is 20-30 ug/ml.

The volume ratio of Sodium Lauryl Ether Sulfate (SLES) solution to DNase I solution is about 10-20: 1.

Preferably, the decellularized lung scaffold is treated with 0.1-1% SLES for 2-3 hours; then, 20-30ug/ml DNase I solution is treated at 25 ℃ for 0.5-1h at a flow rate of 1-2 ml/min.

The 0.1% SLES was used in an amount of 0.6-9ml and the total amount of DNase I solution was about 0.6-3.6 mg.

Specifically, the decellularized lung scaffold is prepared by the following method:

1) firstly, carrying out heparinized PBS (phosphate buffer solution) lavage on the lung scaffold after in vitro treatment;

2) then, lavage is carried out by adopting 0.1% -1% SLES, and lavage is carried out by using deionized water;

3) then, performing lavage treatment by using 20-30ug/ml DNase I solution and performing lavage by using deionized water;

4) finally, lavage with PBS solution containing antibiotics.

Further, the acellular lung scaffold is prepared by sequentially filling the following solutions:

1) heparinized PBS: PBS, phosphate buffer saline (phosphate buffer saline), pH 7.3-7.4; the heparin concentration is 10u/ml, i.e. 1ml PBS added with normal heparin amount is 10u, room temperature, flow rate is 5ml/min, perfusion 15 min. PBS is used within 100 ml.

2) 0.1% -1% SLES: diluting with deionized water, namely adding 0.1-1ml of SLES into every 100ml of deionized water, and perfusing for 2-3h at room temperature and flow rate of 5ml/min, wherein the total dosage of SLES is about 0.6-9 ml. The amount of deionized water was about 600-900 ml.

3) Deionized water: the flow rate was 5ml/min at room temperature and perfusion time was 15 min. The dosage of the deionized water is less than 100 ml.

4) Nuclease application solution: the concentration of the DNase1 liquid is 20-30ug/ml, the DNase1 liquid is diluted by deionized water, namely, 120-30 ug of DNase is added into every 1ml of deionized water, the perfusion temperature is controlled to be 25 ℃, the perfusion flow rate is 1-2ml/min, the perfusion is carried out for 0.5-1h, and the total dosage of the DNase1 is about 0.6-3.6 mg. The amount of deionized water is about 30-120 ml.

5) Deionized water: the flow rate was 5ml/min at room temperature and perfusion time was 15 min. The dosage of the deionized water is less than 100 ml.

6) Antibiotic-containing PBS: penicillin G500U/ml, streptomycin 500ug/ml and amphotericin B25ug/ml, i.e., penicillin G500U, streptomycin 500ug and amphotericin B25ug were added per 1ml PBS. The flow rate was 5ml/min at room temperature and perfusion time was 2 h. PBS was used at about 600ml, and for better washing of cell debris, the perfusion time was extended to 3h and the PBS usage was increased to 1000 ml.

SLES is an anionic detergent used to disrupt cell membranes (lyse cells) to release soluble material within the cell. SLES contains an ethoxy group (C2H5O-), making the chemistry mild. In addition, SLES is readily soluble in water, has excellent detergency, biodegradability and low temperature performance. The lung has loose structure, and the mild SLES is used for decellularizing the lung and can furthest reserve the three-dimensional structure of the lung. At present, the application of SLES to the decellularization of lung is not reported at home and abroad. The investigators of the present invention have experimentally found that SLES infused at a concentration of 0.1% is the minimum concentration for making the SD rat lung nearly transparent, while the three-dimensional structure remains intact.

In order to further remove the cells, the present invention combines perfusion with DNase I solution. DNase I, DeoxyribonucleseI, Chinese name, DNase I, is an endonuclease that digests single-or double-stranded DNA and has the greatest enzymatic activity at 25 ℃. Here, DNase I is used to remove residual DNA, minimizing the possibility of immunological rejection. Earlier researchers have proved through DNA quantitative experiments that after SLES is perfused, the DNA content can be reduced to within 50ng/mg by combining perfusion of DNase I in comparison with non-perfusion of DNase I, so as to meet the requirements of tissue engineering.

The invention provides a preparation method of a decellularized lung scaffold, which comprises the steps of firstly treating for 2-3 hours by 0.1-1% of SLES; then, 20-30ug/ml DNase I solution is treated at 25 ℃ for 0.5-1h at a flow rate of 1-2 ml/min.

The dosage of the SLES is 0.6-9ml, and the total dosage of DNase I solution is about 0.6-3.6 mg.

Specifically, the preparation method of the acellular lung scaffold comprises the following steps:

2) then, lavage is carried out by adopting 0.1-1% SLES, and lavage is carried out by using deionized water;

4) finally, lavage with PBS solution containing antibiotics.

Further, the preparation method of the acellular lung scaffold adopts the following solutions for treatment by sequential perfusion:

1) heparinized PBS: heparin 10u/ml, room temperature, flow rate of 5ml/min, perfusion 15 min.

2) 0.1-1% SLES: diluting with deionized water at room temperature and flow rate of 5ml/min, and perfusing for 2-3h with SLES total dosage of about 0.6-9 ml.

3) Deionized water: the flow rate was 5ml/min at room temperature and perfusion time was 15 min.

4) Nuclease application solution: 20-30ug/ml DNase1 solution is diluted with deionized water, 25 deg.C, flow rate of 1-2ml/min, perfusion time is 0.5-1h, and total dosage is about 0.6-3.6 mg.

5) Deionized water: the flow rate was 5ml/min at room temperature and perfusion time was 15 min.

6) Antibiotic-containing PBS: penicillin G500U/ml, streptomycin 500ug/ml, amphotericin B25ug/ml, room temperature, flow rate of 5ml/min, perfusion for 2 h.

The langendorff perfusion system can control perfusion pressure, flow rate and temperature, ensure the optimal perfusion effect and is beneficial to quantitative production. Researchers apply the scheme to SD rats, the prepared acellular lung scaffold can effectively remove cells, simultaneously perfectly keep the three-dimensional morphological structure of the lung, and extracellular matrix proteins for constructing artificial lungs can be completely stored. The lung stent can be well fused with a receptor after being transplanted in vivo, and has good cellularization and vascularization.

The invention adopts a novel detergent sodium dodecyl ether sulfate (SLES) and a nuclease (DNase1) to solve the problems of serious damage of the three-dimensional structure of the artificial lung and excessive loss of extracellular matrix proteins. By using the langendorff perfusion system, the prepared acellular lung scaffold cells are completely removed, the three-dimensional structure and extracellular matrix proteins are completely reserved, and the allogeneic lung scaffold has good fusibility, cellularization and vascularization after being transplanted subcutaneously for 6 weeks. The preparation method disclosed by the invention is uniform, high in efficiency and simple in steps, can be used for quantitative production, has a good application prospect in the field of tissue engineering lung and loose organs, can be even applied to preparation of decellularized lung of large animals such as pigs and macaques, and provides theoretical basis and technical support for early application of tissue engineering artificial lung to clinic.

Drawings

FIG. 1 is a diagram of the lung status of rats in different perfusion modes; comprising preparing a state diagram of the decellularized lung scaffold at different time points.

FIG. 2 is a graph comparing HE staining of normal rat lung tissue with lung scaffolds prepared according to the present invention;

FIG. 3 is a table showing the DNA content of rat lungs in different perfusion states; DNA content differences between normal rat lungs, decellularized lung scaffolds prepared by perfusion of SLES alone, and decellularized lung scaffolds prepared by combined perfusion of the invention.

FIG. 4 is a comparative scanning electron microscope image of the surface and the inside of a lung tissue of a normal rat and a lung stent prepared by the present invention;

FIG. 5 is a graph of immunofluorescence of normal rat lung versus decellularized lung scaffolds against 5 major extracellular matrix proteins;

FIG. 6 is a graph showing the effect of in vivo transplantation using the decellularized lung scaffold prepared in the present invention.

Detailed Description

The following describes embodiments of the present invention. Elements and features described in one embodiment of the invention may be combined with elements and features shown in one or more other embodiments. It should be noted that the representation and description of components or processes not relevant to the present invention, known to those of ordinary skill in the art, have been omitted for the sake of clarity.

The invention is further described below.

Example 1

First, the lung of the rat is taken

Healthy male clean SD rats are selected, the weight of the SD rats is 250-350g, the SD rats are 8-12 weeks old, the SD rats are fasted for 12 hours before operation, and water is forbidden for 4 hours. Heparin is injected into the abdominal cavity, 1000U/Kg, and after 15min, 3% pentobarbital solution is injected into the abdominal cavity, and the dosage is anaesthetized according to 150 mg/Kg. After the anesthesia was successful, the rat was placed in the supine position and the limb tack was fixed to the operating table. Preparing skin of the operation area of the chest and the abdomen, sterilizing by conventional method, performing combined median incision of the chest and the abdomen with a length of about 5-8cm, cutting diaphragm muscle and rib, opening thymus, and exposing heart and lung. The abdominal viscera was opened with a cotton swab, and the abdominal aorta and the abdominal main vein were cut off to exsanguinate. Injecting 20-50ml of normal saline into the right chamber, wherein each 1ml of normal saline contains 50U of common heparin to prevent blood clot formation in heart and lung, separating and integrally cutting heart and lung bronchus after lung is slightly whitened, and directly storing the obtained fresh normal lung in a refrigerator at-80 ℃ for later use.

Preparation of acellular lung scaffold by Langendorff perfusion method

The normal rat heart lungs previously stored were removed from the-80 ℃ freezer and thawed in a 37 ℃ water bath. The lungs were slightly inflated by injecting 20ml of PBS solution through the main bronchi, containing 100U of plain heparin. Finding and separating pulmonary vein and main pulmonary artery along trachea and ascending aorta, separating heart and ascending aorta via main pulmonary artery root, inserting 16G perfusion head into main pulmonary artery, and perfusing lung residual blood with heparinized PBS solution. And (3) suspending the perfusion needle head to a langendorff operating platform, adjusting the perfusion needle head to reach a proper distance with the heat preservation chamber, and performing isolated lung perfusion through the pulmonary artery. Maintaining the perfusion pressure at 20cmH in the 1 st, 2 nd, 3 th, 5 th and 6 th steps₂O, room temperature, flow rate 5 ml/min. Maintaining perfusion in step 4Pressure 20cmH₂O, the temperature is 25 ℃, and the flow rate is 1-2 ml/min.

The following solutions were sequentially poured:

1. heparinized PBS: heparin 10u/ml, room temperature, flow rate of 5ml/min, perfusion 15 min.

2.0.1% SLES: diluting with deionized water at room temperature and flow rate of 5ml/min, and perfusing for 2h to obtain SLES total dosage of about 0.6 ml.

3. Deionized water: the flow rate was 5ml/min at room temperature and perfusion time was 15 min.

4. Nuclease application solution: 30ug/ml DNase1 solution was diluted with deionized water at 25 deg.C and flow rate of 2ml/min, and perfused for 1h, with a total dose of about 3.6 mg.

Most of the existing methods are soaked in 30ug/ml DNase1 solution for 0.5-1 h. The DNase1 perfusion method adopted by the invention can remove DNA more effectively. However, if too much DNase1 is used, the damage to the three-dimensional structure is serious, and the flow rate of 1-2ml/min is preferably used in the present invention.

5. Deionized water: the flow rate was 5ml/min at room temperature and perfusion time was 15 min.

6. Antibiotic-containing PBS: penicillin G500U/ml, streptomycin 500ug/ml, amphotericin B25ug/ml, room temperature, flow rate of 5ml/min, perfusion for 2 h. The purpose of this step is to wash out residual cell debris and detergent. The step does not have uniform perfusion time at present, the invention adopts perfusion for 2 hours, rejection reaction does not occur after in vivo transplantation, and the cellularization and vascularization are good.

Experimental example 1

The lung status of the rat was observed by different perfusion methods, as shown in fig. 1, a is normal rat lung taken out of the refrigerator, separated from the heart, inserted into the 16G perfusion head via the main pulmonary artery, and suspended to the langendorff console for waiting perfusion. B is perfused with 0.1% SLES solution at a flow rate of 5ml/min, and after 2 hours the lungs are substantially clear, and to achieve the effect of substantial transparency, the perfusion time can be extended to 3 hours. C is the flow rate of 2ml/min after the DNase I solution with the perfusion concentration of 30ug/ml is filled for 1 hour, so that the lung is more glittering and translucent. And D, obtaining the acellular lung scaffold prepared by the invention after the final completion of perfusion. After the stent prepared by the method is infused with the methylene blue dye solution, researchers can see that the blue dye solution is uniformly distributed, and the capillary bed is completely reserved. It can be concluded from fig. 1 that the decellularized lung scaffold prepared by the present invention can completely retain the three-dimensional structure and the microcirculation structure.

Experimental example 2

FIG. 2 is a graph comparing HE staining of normal rat lung tissue with lung scaffolds prepared according to the present invention; and (3) randomly taking normal lung lobes and lung lobes of the decellularized lung stent according to the conventional HE staining steps, namely fixing, dehydrating, embedding, preparing slices and performing conventional HE staining. As shown in FIG. 2, A is HE staining of lung tissue of normal rat, and the tissue is full of cells; b is the lung scaffold HE staining prepared by the invention, the tissue has no obvious cell distribution, the magnification is 100 times, and the scale is 100 um. It can be concluded from FIG. 2 that the method of the present invention can completely remove normal lung cells.

Experimental example 3

FIG. 3 is a chart of DNA content in rat lungs from different perfusion states to evaluate the effectiveness of the combination of DNase I. We randomly divided 18 rats into 3 groups, 6 normal lung groups, 6 DNase I-groups, and 6 DNase I + groups. The normal lung group was the lungs taken out of the refrigerator without any perfusion. DNase I-group was perfused with SLES only, and not DNase I. DNase I + group the combined perfusion SLES and DNase I method of the invention was used. The lung tissues obtained by the three groups of methods were subjected to DNA content determination according to the QIAamp DNA MiniKit kit instructions. Data were analyzed for one-way anova using SPSS software. The results are shown in FIG. 3. Normal lung group: 1113.7 ± 56.9 (mean ± standard deviation); DNase I-group: 63.9 +/-7.9; 33.9. + -. 3.1 for DNase I +. Unit: ng/mg (dry weight). Compared with normal lung tissue, the DNA content is obviously reduced after decellularization (p is less than 0.05), although there is no statistical difference between the DNase I-group and the DNase I + group (p is more than 0.05), the DNA content of the DNase I + group is within 50ng/mg, namely the DNA content in each 1mg of decellularized scaffold tissue is not more than 50 ng. It can be concluded from FIG. 3 that the present invention can remove 97% of DNA content in normal lung, and meet the requirements of tissue engineering.

Experimental example 4

FIG. 4 is a comparative scanning electron microscope image of the surface and the inside of a lung tissue of a normal rat and a lung stent prepared by the present invention; (ii) a The scanning electron microscope was manufactured according to the conventional operation procedure, and the results are shown in fig. 4, using the scanning electron microscope: a Normal rat Lung surface ultrastructure. B is the ultramicro structure on the surface of the acellular lung scaffold, which is basically similar to the normal lung and is smooth and complete. C is the inner ultrastructure of normal rat lung and is full of cells. D is an ultramicro structure in the decellularized lung scaffold, no cell residue is seen, and the three-dimensional structure is basically close to that of a normal lung. A. B is enlarged by 2000 times and has a scale bar of 50 um. C. D is enlarged by 1000 times and has a scale bar of 100 um. It can be concluded from fig. 4 that the ultrastructure of the decellularized lung scaffold prepared by the present invention also remains intact, close to normal lung tissue.

Experimental example 5

FIG. 5 is a graph of immunofluorescence of normal rat lung versus decellularized scaffold against 5 extracellular matrix proteins; normal lung tissue and the acellular lung scaffold tissue prepared by the invention are prepared into paraffin sections according to a conventional immunofluorescence process, and the paraffin sections are subjected to antigen retrieval, goat serum sealing, primary antibody incubation, addition of corresponding fluorescent secondary antibody and observation under a fluorescence microscope. As shown in fig. 5, the decellularized lung scaffold of the present invention can substantially perfectly retain 5 major extracellular matrix proteins: type IV collagen, type I collagen, fibroblast, laminin and elastin, the fluorescence expression level is basically close to that of normal lung. Magnification is 200 times, scale bar 75 um. It can be concluded from fig. 5 that 5 key proteins constituting the extracellular matrix are well preserved, which provides a good host environment for in vivo transplantation of cells and vascularization.

Experimental example 6

FIG. 6 is a graph showing the effect of in vivo transplantation treatment using the decellularized scaffold of the invention; the right upper lung lobe of the decellularized lung scaffold prepared by the inventor is about 2cm multiplied by 1.5cm in size. The receptor SD rat is anesthetized and disinfected, the abdominal skin is cut, the upper right lung lobe of the decellularized lung stent is transplanted to the abdominal subcutaneous part of the receptor rat, the suturing and disinfection are carried out, the whole process is carried out by aseptic operation, and the receptor SD rat is bred conventionally after the operation. Implanted tissue was removed after 6 weeks for modified Masson staining. The results are shown in FIG. 6, where A is the transplantation of the decellularized lung scaffold into the subcutaneous tissue of another SD rat for 6 weeks, it can be seen that the scaffold is well fused with the surrounding tissue, and there is no rejection such as necrosis and exfoliation. B is a modified Masson trichrome stain showing neovascularization (arrow) and cellular infiltration (red) around it. It can be concluded from fig. 6 that the acellular lung scaffold prepared by the invention has good histocompatibility and good cellularization and vascularization effects. This provides the experimental basis for final organ transplantation in situ and long-term survival.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, devices, means, methods, or steps.

Claims

1. A preparation method of a decellularized lung scaffold is characterized in that the decellularized lung scaffold is prepared by the following method:

1) firstly, the lung scaffold after in vitro treatment is subjected to heparinized PBS solution lavage: perfusing heparinized PBS with heparin concentration of 10U/mL, room temperature, flow rate of 5mL/min, perfusing for 15 min;

2) then, lavage is carried out by adopting sodium dodecyl ether sulfate with the volume percentage concentration of 0.1-1%, and lavage is carried out by using deionized water: diluting sodium dodecyl ether sulfate with deionized water, namely adding 0.1-1mL of SLES into every 100mL of deionized water, and perfusing for 2-3h at room temperature and flow rate of 5 mL/min; then deionized water is used for perfusion for 15min at room temperature and the flow rate of 5 mL/min;

3) subsequently, lavage treatment was performed with 20-30ug/mL DNase I solution and with deionized water: diluting DNase I with deionized water, namely adding 120-30 ug of DNase into every 1mL of deionized water, controlling the perfusion temperature to be 25 ℃, controlling the perfusion flow rate to be 1-2mL/min, and perfusing for 0.5-1 h; then deionized water is used for perfusion for 15min at room temperature and the flow rate of 5 mL/min;

4) final lavage with PBS solution containing antibiotics: penicillin G500U, streptomycin 500ug and amphotericin B25ug were added per 1mL PBS at room temperature and flow rate of 5mL/min for 2h of perfusion.