CN104208751A - Preparation method for novel kidney acellularized biological scaffold - Google Patents

Abstract

A preparation method of a novel kidney decellularized bioscaffold does not use the conventionally used ionic detergent SDS, which has a destructive effect on the scaffold structure, but uses Triton-100 solution containing sodium carbonate as the detergent. There will be no adverse effects of the ionic detergent SDS destroying the scaffold structure, and sodium carbonate, as an alkaline substance, can enhance the effect of decellularization by adding an appropriate amount.

Description

Preparation method of a novel kidney decellularized bioscaffold

technical field

The invention relates to the field of organ tissue decellularization, in particular to a preparation method of a novel kidney decellularized biological scaffold. the

Background technique

Tissue engineering scaffold materials are an important basis for constructing tissue engineered organs or tissues. Extracellular matrix (ECM) is not only a good biological scaffold, but also an important signal regulator molecule for cells, which can regulate cell growth, metabolism and differentiation. It has the macroscopic and microscopic structure of the original tissue, and is rich in extracellular matrix components left after decellularization, such as collagen, elastic fibers, laminin, and fibronectin. Extracellular matrices obtained from different tissues, including skin, blood vessels, nerves, bones, tendons, small intestinal submucosa, bladder, heart valves, etc., have been extensively studied for renewable material applications. In recent years, products using ECM as a scaffold material and decellularization technology have been applied clinically, including human skin, intestinal submucosa (SIS), porcine bladder and porcine heart valves, etc. With the development of tissue engineering, the decellularization technology of whole organs has gradually become one of the research hotspots of scholars in related fields. Ott et al. used the isolated mouse heart to retain the ECM of the heart after decellularization technology, reinjected newborn mouse cells, and restored the beating of the heart after in vitro culture, making it possible to reconstruct physiologically active organs in vitro. Organ transplants offer new hope. However, due to the special structure and complex functions of the kidney, the research on its cell scaffolds is still mainly focused on artificially synthesized scaffolds, and there are few reports on the whole kidney extracellular matrix as a biological scaffold. In 2009, my country Liu Chunxiao et al. successfully prepared rat whole kidney acellular extracellular matrix by detergent perfusion; in 2010, Nakayama et al. used 1% sodium dodecyl sulfate (SDS) or 1% Triton X -100) soaking method to prepare extracellular matrix of macaque kidney. At present, the preparation methods of ECM at home and abroad are becoming more and more mature, and there are two main categories: one is enzymatic method: commonly used enzymes include trypsin, deoxyribonuclease and ribonuclease; the other is chemical method: commonly used detergents are SDS , Triton X-100, Triton X-200, sodium deoxycholate, etc. However, it has been reported that trypsin at a normal concentration (0.25%) can hydrolyze collagen and elastin, and destroy the structure of the valve stent. The detergent SDS has a destructive effect on the scaffold structure. the

Contents of the invention

In order to solve the shortcomings of the prior art, the present invention provides a preparation method for designing and synthesizing a novel kidney decellularized bioscaffold, using Triton-100 solution containing sodium carbonate as a detergent to prepare the kidney decellularized bioscaffold. the

A preparation method of a novel kidney decellularized bioscaffold, the preparation method comprising the following steps:

(1) Intubate the artery of the isolated kidney with a 22G indwelling needle, and wash the residual blood in the kidney with heparin and PBS until the kidney is uniformly khaki;

(2) Perfuse the whole kidney tissue with 2000ml 0.1mol/L sodium carbonate and 1% Triton x-100 mixed solution dissolved in PBS and 500ml mixed solution of 4% ethanol and 0.01% peracetic acid at a constant temperature of 37°C. And the PBS solution containing penicillin and streptomycin, and then continue to perfuse, soak the kidney tissue in the perfusion solution at the same time, the perfusion speed is 15ml/min, observe the changes in the color and shape of the kidney during the perfusion process, and wait until the color of the kidney changes. Light, from the original khaki to white and transparent. The kidney decellularized scaffold was obtained, and the prepared kidney decellularized scaffold was stored at -80°C.

In the preparation method of a novel kidney decellularized bioscaffold, the perfusion pressure during perfusion in the step (2) is 3.6 mmHg. the

In the preparation method of a novel kidney decellularized bioscaffold, the concentration of the PBS solution containing penicillin and streptomycin in the step (2) is 105 IU/L. the

In the preparation method of a novel kidney decellularized bioscaffold, the infusion time of the PBS solution containing penicillin and streptomycin in the step (2) is 8-10 h

The beneficial effect of the present invention is: the present invention provides a kind of preparation method of novel kidney decellularized bioscaffold, does not use the ionic detergent SDS that conventionally uses and has damaging effect to scaffold structure, but adopts the preparation method that contains sodium carbonate The Triton-100 solution of Triton-100 solution is used as a detergent, which will not cause the adverse effect of the ionic detergent SDS on destroying the scaffold structure, and sodium carbonate is used as an alkaline substance, adding an appropriate amount can enhance the effect of decellularization.

Description of drawings

Fig. 1 is a graph showing changes in color and shape of the whole kidney of the present invention. the

Figure 2 is the transmission electron microscope observation of the renal decellularized scaffold and the kidney of the control group. the

Fig. 3 is the HE, PAS and Masson observation images of the renal decellularized scaffold and the kidney of the control group. the

Fig. 4 is a picture of protein immunofluorescence analysis of renal decellularized scaffolds and kidneys of the control group. the

Figure 5 is a diagram showing the shape and distribution of blood vessels in the kidney under a stereo microscope. the

In Figure 2, 1-podocytes, 2-foot processes, 3-basement membrane, 4-vascular endothelial cells, 5-microvilli, 6-tubular membrane, 7-mitochondria, 8-interstitial cells, 9-collagen fibers, 10- glomerulus, 11- capillary membrane, 12- mesangium. the

the

Detailed ways

Preparation of isolated rat kidney

Take healthy adult SD rats, weigh them, anesthetize by intraperitoneal injection of 0.3-0.4ml/100g 10% chloral hydrate, fix them after the effect, open the abdominal cavity, separate the inferior vena cava, inject heparin sodium solution for whole-body heparinization . Bluntly dissect the bilateral kidneys, and the kidneys are brownish-red. Peel off the three layers of capsules of the kidneys, find the renal pedicle, separate the renal artery, vein and ureter, cut off the ureter, ligate and cut off the beginning of the renal artery and vein, Kidneys were taken, one side of the kidney was perfused and decellularized, and the other side of the kidney was stored at -80°C as a control group.

Decellularization of kidney tissue

(1) One kidney tissue was stored at -80°C without further processing as a control group.

(2) Kidney decellularization group: the arterial 22G indwelling needle of the isolated kidney was intubated, and the residual blood in the kidney was washed with heparin and PBS until the kidney was uniformly khaki. Perfuse the whole kidney tissue with 2000ml 0.1mol/L sodium carbonate and 1% Triton x-100 mixed solution dissolved in PBS, and 500ml mixed solution of 4% ethanol and 0.01% peracetic acid at a constant temperature of 37°C, and cyanide containing The PBS solution containing penicillin and streptomycin, the perfusion time of the PBS solution containing penicillin and streptomycin is 8-10 h, the concentration of the PBS solution containing penicillin and streptomycin is 105 IU/L, and then the perfusion is continued. Soak the kidney tissue in the perfusion solution. The perfusion pressure during perfusion is 3.6mmHg, and the perfusion rate is 15ml/min. During the perfusion process, observe the changes in the color and shape of the kidney. White and transparent. The kidney decellularized scaffold was obtained, and the prepared kidney decellularized scaffold was stored at -80°C. the

Renal tissue decellularization data analysis and processing

1. General observation:

Observe the changes in the color and shape of the fresh kidney and the whole kidney during the whole process of perfusion and immersion, as shown in Figure 1: the kidney tissue changes from brown-red to uniform khaki, and then gradually turns white until it is completely transparent.

2. Genomic DNA content analysis:

Take 50-80 mg of cortex and medulla from the kidney of the control group and the kidney of the decellularized group respectively, after homogenization, cell lysis, protein removal, and purification, genomic DNA is extracted (operated according to the instructions of the DNA kit), and measured by an ultraviolet spectrophotometer. After its concentration, parallel agarose gel electrophoresis for its qualitative determination.

The results are shown in Table 1 below:

It can be seen from Table 1

The renal scaffolds of the control group and the decellularized group were tested for DNA. The results showed that the average DNA content in the renal cortex of the control group was 4.95×10 ^-4 ng/L, and the average DNA content in the medulla was 3.48×10 ^-4 ng/L; the average DNA content of renal cortex in decellularized group 1 was 4.67×10 ^-6 ng/L, the average DNA content in medulla was 9.81×10 ^-6 ng/L, and the proportion of decellularized cells could be as high as More than 97%. It shows that the DNA content of the decellularized group has been greatly reduced.

3. Transmission electron microscope observation:

The kidneys of the control group and the decellularized group were cut into 1.0 mm3 tissue blocks respectively, and fixed in 2.5% glutaraldehyde at 4°C for 12 hours. After washing with PBS, fix with osmium acid, stain with uranyl acetate, dehydrate with gradient acetone, soak with epoxy resin embedding agent, embed, make ultra-thin sections, and observe under transmission electron microscope.

as shown in picture 2:

Transmission electron microscope observation of kidney decellularized scaffold and control group kidney in Figure 2

A-C normal kidney; D-F decellularized kidney; in Figure 2 A, 1 is podocytes, 2 is foot process, 3 is basement membrane, 4 is vascular endothelial cells; in Figure 2 B, 5 is microvilli, 6 is tubular membrane , 7 is mitochondria; in Figure 2C, 8 is interstitial cells, and 9 is collagen fibers; in Figure 2D, 10 is glomerulus, 11 is capillary membrane, and 12 is mesangium; in Figure 2E, 6 is Renal tubule membrane; 9 in F in Fig. 2 is collagen fiber, 6 is renal tubule membrane, and 11 is capillary membrane.

In the renal cortex of the control group, the structure of the renal corpuscle was complete and clear, and the tubules adjacent to the renal corpuscle were denser and difficult to distinguish; a large number of tubular structures were seen in the medulla, the thicker tubules were the proximal convoluted tubules, and the thinner tubules were the distal convoluted tubules. Tubules and collecting ducts. In the decellularized group, the outline structure of glomeruli and renal tubules was basically complete, and the extracellular matrix such as basement membrane and fibrin was scattered. "Geographic" structure, the lumen collapsed, hollow without cell residues. the

4. Dyeing observation:

Figure 3 is the HE, PAS and Masson observations of the renal decellularized scaffolds and the kidneys of the control group

In Figure 3, the top is the normal kidney; the bottom is the decellularized kidney scaffold; a large number of cells can be seen in the normal kidney; there is no obvious cell residue in the decellularized kidney, the structure of the glomeruli and renal tubules is intact, and the glycogen components and fibers are fully preserved.

5. Immunofluorescence detection:

Nine paraffin sections were randomly selected from the kidneys of the control group and the decellularized group. Immunofluorescence was used to detect the expression of collagen IV, LN and FN. Under the fluorescence microscope, the nuclei showed blue fluorescence and the basement membrane showed green fluorescence, indicating positive expression.

Figure 4 is the protein immunofluorescence analysis of renal decellularized scaffolds and kidneys of the control group

Upper: normal kidney; lower: decellularized kidney scaffold; in the picture, a large number of blue-stained nuclei in the normal kidney; no obvious nucleus remains in the decellularized kidney matrix, and collagen is fully preserved.

6. Observation of renal artery casts:

Perfuse the renal artery with 2-4ml of 10% acrylonitrile-butadiene-styrene resin (ABS) Sudan red solvent, maintain a certain pressure during perfusion, rinse and cool with running water, corrode with 50% hydrochloric acid for 1-3 days, rinse with clean water, and use a stereomicroscope Observe the shape and distribution of blood vessels in the kidney.

Figure 5 is the morphology and distribution of intrarenal blood vessels under a stereo microscope

Among them, A is the kidney of the control group, and B is the decellularized kidney. It can be seen from Figure 5 that the blood vessels in the decellularized group are distributed in a "branch-like" manner, which is relatively sparse, and the branches of the blood vessels are still clearly visible and the shape is complete.

The preparation method of the kidney decellularized bioscaffold in this embodiment can also be used to prepare human or other animal kidney decellularized bioscaffolds, such as cadaveric kidney, pig kidney, monkey kidney, etc. the

Claims (4)

1. a preparation method for Novel kidney acellular organism support, is characterized in that, described preparation method comprises the following steps:

(1) in the tremulous pulse 22G remaining needle intubate of Dispersing Monkey Kidney Cell in Tissue Culture, rinsing remained blood in kidney with heparin PBS, is homogeneous khaki to kidney;

(2) whole renal tissue is poured at constant temperature 37 DEG C the Triton x-100 mixed solution that 2000ml 0.1mol/L sodium carbonate and 1% is dissolved in PBS successively, and 4% ethanol of 500ml and 0.01% peracetic acid mixed solution, and containing blue or green, the PBS solution of streptomycin, then Continuous Perfusion, while perfusion, nephridial tissue is soaked in infusion liquid, rate of flooding is 15ml/min, the change of kidney color and form is observed in filling process, treat kidney lighter, white is become and transparence from original khaki, obtain kidney Acellularized valve, obtained kidney Acellularized valve is stored in-80 DEG C.

2. the preparation method of a kind of Novel kidney acellular organism support according to claim 1, is characterized in that, the perfusion pressure in described step (2) during perfusion is 3.6mmHg.

3. the preparation method of a kind of Novel kidney acellular organism support according to claim 1, is characterized in that, the PBS solution concentration containing penicillin and streptomycin in described step (2) is 105 IU/L.

4. the preparation method of a kind of Novel kidney acellular organism support according to claim 1, is characterized in that, in described step (2), perfusion is 8-10 h containing the infusion time of the PBS solution of penicillin and streptomycin.