Tissue gel, preparation method thereof and application of tissue gel in preparation of medicine for treating adrenal insufficiency

Abstract

The invention belongs to the technical field of tissue engineering, and particularly relates to tissue gel, a preparation method thereof and application of the tissue gel in preparation of drugs for treating adrenal insufficiency. According to the tissue gel, adrenocorticotropin and glucose are dispersed in a hydrogel matrix material, and the mass ratio of the hydrogel matrix material to the adrenocorticotropin to the glucose is (2.5-5): (3 * 10 <-6 >-120 * 10 <-6 >): 50. According to the invention, the unique property of hyaluronic acid is utilized, hyaluronic acid is used as a gel matrix material, the composite tissue gel is formed by adding adrenocorticotropin into hyaluronic acid, and glucose can provide enough energy supply for tissues. The composite tissue gel is used for treating the adrenal gland tissue, activity maintenance before transplantation can be well provided for the adrenal gland tissue, and compared with a traditional transplantation donor treatment method, the survival rate of the transplanted tissue can be increased to a certain degree through treatment of the composite tissue gel.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue engineering, and in particular, relates to a tissue gel and a preparation method thereof and application of the tissue gel in preparing a medicine for treating adrenal insufficiency. Background Art

[0002] Lifelong use of glucocorticoids is the mainstream effective means of treating adrenal insufficiency. However, long-term medication will bring a series of irreversible and very serious side effects to patients. Therefore, the clinical demand for the development of new treatment methods is very urgent. Cell engineering is an emerging ideal treatment method to replace long-term medication. However, due to the relatively single cell type and the limitation of the volume and number of cell transplants, the long-term stable function of the transplant is not ideal. Therefore, there has been no substantial breakthrough in the long-term treatment of adrenal insufficiency. At the same time, due to the lack of relatively complete tissue morphology, the precise and rich vascular network necessary for the function of tissues and organs cannot be formed in the transplant.

[0003] The existing large tissues and organs in the body have a highly organized mature vascular system, which can provide a sufficient vascular network for the regeneration of tissues and organs in the host. The spleen is currently considered to be an ideal place for tissue regeneration in the host due to its unique structure. First of all, the spleen itself mainly plays the immune function of lymphatic organs and the filtering function of the blood system, which can be compensated to a large extent by the abundant lymph node tissues and liver and kidneys in the body. Secondly, existing clinical data show that after the spleen is damaged and removed, the health status and life expectancy of a large proportion of patients can be maintained at normal levels, but it cannot be ruled out that a small proportion of patients may develop infections due to immune problems, but after proper rehabilitation treatment, the long-term survival status of patients can still remain relatively stable.

[0004] The spleen's natural structure is relatively loose, and its internal space is very ample compared to the adrenal tissue. There is sufficient space inside to accommodate the transplanted adrenal tissue well, and it has little impact on its own function after transplantation.

[0005] Currently, adrenal insufficiency can be treated by transplanting adrenal tissue into the spleen. However, adrenal tissue isolated from the body is easily inactivated, and the tissue regeneration rate after transplantation is limited. Therefore, it is very necessary to provide a tissue gel and a preparation method thereof, which can wrap and protect adrenal tissue before transplantation, thereby promoting the regeneration of adrenal tissue in vivo. Summary of the invention

[0006] The primary purpose of the present invention is to provide a tissue gel and a preparation method thereof and use thereof in the preparation of a drug for treating adrenal insufficiency, so as to achieve the treatment of adrenal tissue before transplantation, provide good activity maintenance for adrenal tissue before transplantation, and improve the survival rate of transplanted tissue.

[0007] To this end, the present invention provides the following technical solutions.

[0008] The first aspect of the present invention provides a tissue gel, wherein adrenocorticotropic hormone and glucose are dispersed in a hydrogel matrix material, and the mass ratio of the hydrogel matrix material, the adrenocorticotropic hormone and the glucose is (2.5-5): (3×10 -6 ~120×10 -6 ):50.

[0009] In some embodiments, the hydrogel matrix material is hyaluronic acid.

[0010] The second aspect of the present invention provides a method for preparing the tissue gel as described above, the method comprising the following steps:

[0011] S1: dissolving glucose and ACTH in water to obtain ACTH solution;

[0012] S2: adding the hydrogel matrix material to the ACTH solution, mixing and dissolving to obtain a tissue gel solution.

[0013] In some embodiments, the concentration of the ACTH solution in step S1 is 3 to 120 ng / L, and suitable concentrations may be, for example, 3 ng / L, 5 ng / L, 10 ng / L, 20 ng / L, 30 ng / L, 40 ng / L, 50 ng / L, 60 ng / L, 70 ng / L, 80 ng / L, 90 ng / L, 100 ng / L, 110 ng / L, and 120 ng / L.

[0014] In some embodiments, the concentration of glucose in step S1 is 50 mg / mL.

[0015] In some embodiments, the concentration of the hyaluronic acid in step S2 is 2.5-5 mg / mL, and suitable concentrations may be, for example, 2.5 mg / mL, 2.75 mg / mL, 3 mg / mL, 3.25 mg / mL, 3.5 mg / mL, 3.75 mg / mL, 4 mg / mL, 4.25 mg / mL, 4.75 mg / mL, and 5 mg / mL.

[0016] In a third aspect of the present invention, there is provided a use of the tissue gel as described above in the preparation of a medicament for treating adrenal insufficiency.

[0017] The fourth aspect of the present invention provides a drug for treating adrenal insufficiency, comprising: the tissue gel as described above, and adrenal tissue.

[0018] In some embodiments, the adrenal insufficiency is secondary adrenal insufficiency and / or primary adrenal insufficiency.

[0019] In some embodiments, the adrenal insufficiency comprises congenital adrenal hyperplasia, unilateral or bilateral adrenalectomy, and iatrogenic Cushing's syndrome.

[0020] In some embodiments, the congenital adrenal hyperplasia includes 21-hydroxylase deficiency syndrome and 17-hydroxylase deficiency syndrome.

[0021] A fifth aspect of the present invention provides a method for preparing the above-mentioned drug, the method comprising the following steps:

[0022] i) cutting the adrenal tissue along the midline to obtain an adrenal tissue block;

[0023] ii) The adrenal tissue block is premixed in the tissue gel solution as described above, and then taken out and allowed to stand and solidify to obtain the adrenal tissue gel.

[0024] In some embodiments, the adrenal tissue in step i) can be cut into adrenal tissue blocks of different volumes, for example, 1 / 2 volume, 1 / 4 volume, 0.7 mm volume, 0.5 mm volume, and 0.2 mm volume.

[0025] In some embodiments, the premixing conditions in step ii) are: temperature 37° C., time 10 to 15 min.

[0026] By means of the above technical solution, the present invention has at least the following advantages:

[0027] The present invention utilizes the unique properties of hyaluronic acid itself, takes hyaluronic acid as gel matrix material, and forms a composite tissue gel by adding adrenocorticotropic hormone and glucose to hyaluronic acid. The composite tissue gel is used to treat adrenal tissue, wherein glucose can provide sufficient energy supply for the tissue, and can provide adrenal tissue with good activity maintenance before transplantation. Compared with the traditional transplant donor treatment method, the composite tissue gel treatment of the present invention can improve the survival rate of transplanted tissue to a certain extent. Furthermore, this new tissue gel not only realizes the successful transplantation of adrenal tissue, but also retains the function of the spleen itself to the greatest extent. The mature vascular network system inside the spleen can help adrenal tissue to complete the reconstruction of its own vascular network and the function of the transplantation more quickly. The present invention uses tissue gel to treat adrenal tissue before transplantation, and at the same time develops the spleen as a new site for adrenal tissue transplantation, which provides a new treatment strategy for the in vivo regeneration of the adrenal gland and the treatment of adrenal insufficiency, and provides a research direction for the development and preparation of drugs for the treatment of adrenal insufficiency, which has a very realistic clinical significance.

[0028] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a micrograph of mouse adrenal tissue after pretreatment with tissue gel before transplantation;

[0030] Figure 2 HE histology was used to detect the structural changes of adrenal tissue after transplantation;

[0031] Figure 3 Immunofluorescence was used to detect the expression of CYP21 cortisol catalyzing enzyme in adrenal tissue at different time points;

[0032] Figure 4 The changes in the number of lymphocytes in the blood of mice after transplantation of adrenal tissue gel;

[0033] Figure 5 This is the change of cortisol and ACTH levels in the serum of mice after adrenal tissue gel transplantation. DETAILED DESCRIPTION

[0034] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0036] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0037] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Example 1: Preparation of tissue gel solution

[0041] Dissolve glucose and adrenocorticotropic hormone in 1L of double distilled water in turn to obtain adrenocorticotropic hormone solutions, in which the concentrations of adrenocorticotropic hormone are 3ng / L, 5ng / L, 10ng / L, 20ng / L, 30ng / L, 40ng / L, 50ng / L, 60ng / L, 70ng / L, 80ng / L, 90ng / L, 100ng / L, 110ng / L, and 120ng / L, and the concentration of glucose is 5%.

[0042] 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, 3.75 mg, 4 mg, 4.25 mg, 4.75 mg, and 5 mg of hyaluronic acid (9002-60-2, MCE) were respectively added to 1 mL of the above-mentioned adrenocorticotropic hormone solution, and the mixture was mixed and dissolved to obtain tissue gel solutions of different specifications.

[0043] Comparative Example 1: Preparation of gel solution

[0044] The only difference from Example 1 is that adrenocorticotropic hormone is not added, and the rest is the same as Example 1 to obtain a gel solution.

[0045] Example 2: Acquisition of adrenal tissue

[0046] In this example, mice were used as subjects to obtain mouse adrenal tissues. The specific operation method was as follows:

[0047] The donor mice (purchased from Jicui Yaokang Biotechnology Co., Ltd.) were killed by cervical dislocation. The mouse abdomen was fully wetted with 75% ethanol to disinfect the mouse, and the outer skin and endothelium were cut along the midline of the mouse abdomen with surgical scissors to fully expose the mouse abdominal cavity. The mouse intestinal tissue was gently pushed aside to expose the bilateral kidneys of the mouse, and the adrenal tissue was gently separated from the kidneys with micro-ophthalmic forceps to maintain a small distance. The adrenal gland was gently cut off from the side close to the kidney with ophthalmic scissors. During the cutting process, excessive residue of adipose tissue and rupture of adrenal veins should be avoided as much as possible to obtain the separated adrenal tissue. The separated adrenal tissue was placed in a pre-cooled PBS solution and washed repeatedly for 3-5 times to remove blood stains attached to the surface. After that, the residual fat and connective tissue attached to the adrenal tissue were carefully removed with ophthalmic scissors, and rinsed with pre-cooled PBS buffer solution for 3 to 5 times. Finally, it was placed in a PBS solution for use.

[0048] Example 3: Treatment of adrenal tissue

[0049] The adrenal tissue obtained in Example 2 was cut using microscopic ophthalmic scissors under a microscope, and the cutting was carried out in a step-by-step manner, that is, the tissue was quickly cut along the midline of the adrenal tissue, and unnecessary damage to the tissue was avoided as much as possible during the cutting process to avoid affecting subsequent transplantation, and finally adrenal tissue blocks of 1 / 2 volume, 1 / 4 volume, 0.7mm volume, 0.5mm volume, and 0.2mm volume were obtained. The adrenal tissue blocks of different volumes were rinsed 1 to 2 times in a precooled PBS buffer solution, and then each adrenal tissue block was transferred to a sterile culture dish containing precooled PBS for standby use. The tissue gel solutions of different specifications obtained in Example 1 were premixed with the adrenal tissues of different volumes obtained at 37°C for 15 minutes, so that the tissue gel solutions were fully contacted and the adrenal tissues were completely wrapped, and then taken out and allowed to stand for solidification to obtain adrenal tissues wrapped with tissue gels of different specifications, hereinafter referred to as adrenal tissue gels.

[0050] According to the above method, the adrenal tissues were divided into two parts, 1 / 2 volume adrenal tissue blocks, as the experimental group and the control group, one part for each group, and the adrenal tissue blocks of each group were processed as follows:

[0051] Experimental group: adrenal tissue blocks were added to the tissue gel solution obtained in Example 1 (ACTH concentration of 70 ng / L, HA concentration of 3.5 mg / L) and premixed at 37°C for 15 min, then taken out and allowed to stand and solidify to obtain adrenal tissue gel.

[0052] Control group: adrenal tissue pieces without any treatment.

[0053] The adrenal tissue gel and adrenal tissue blocks obtained from the experimental group and the control group were observed under a microscope. Figure 1 .like Figure 1 As shown, the left picture is the experimental group and the right picture is the control group. It can be clearly observed under the microscope that the adrenal tissue gel obtained after pre-treatment of the adrenal tissue before transplantation with tissue gel solution has a complete structure and clear tissue contours, and there is no obvious tissue structure damage or even loose tissue.

[0054] Example 4: Transplantation of adrenal tissue gel

[0055] In this embodiment, mice were used as the subjects, and adrenal tissue was transplanted into the spleen of mice. The specific operation method was as follows: a 1 cm long incision was made near the left rib of the mouse (purchased from Jicui Yaokang Biotechnology Co., Ltd.) to expose the mouse endothelium, and then a 0.5 cm long incision was added to the exposed mouse endothelium to expose the mouse abdominal cavity. The mouse spleen and blood vessels were gently pulled out of the abdominal cavity from the endothelial opening to between the endothelium and the outer skin. The cut mouse endothelium was sutured to prevent the spleen from retracting into the abdominal cavity. Suture the mouse outer skin. After the suture is completed, gently press the suture to adjust the displaced spleen to the optimal position. Allow the mice to recover for one week after the operation in preparation for the implementation of further adrenal transplantation surgery. Thus, the mice to be transplanted were obtained.

[0056] The mice to be transplanted were divided into three groups: experimental group, gel group and control group. First, a 1 cm long incision was made on the skin where the spleen of the above-mentioned mice to be transplanted was located to expose the displaced spleen. After that, the mice in each group were treated as follows:

[0057] Experimental group: Adrenal tissue gel (adrenal glands with a volume of 1 / 2 of the volume of the tissue gel of Example 1, wherein the hyaluronic acid concentration in the tissue gel is 3.5 mg / L and the ATCH concentration is 70 ng / L) is selected according to the demand and moved into the spleen with fiber ophthalmic forceps to complete the transplantation of the adrenal tissue gel. After the transplantation is completed, it is left to stand for a while and when there is no obvious blood outflow from the surface of the spleen, the epidermis is sutured to complete the transplantation operation.

[0058] Gel group: Adrenal tissue gel (the gel of comparative example 1 was used to wrap 1 / 2 volume of adrenal gland, wherein the concentration of hyaluronic acid in the gel was 3.5 mg / L) was used for mouse spleen transplantation in the same manner as the experimental group.

[0059] Control group: Adrenal tissue blocks (1 / 2 the volume of the adrenal gland) were transplanted into mouse spleens in the same manner as in the experimental group.

[0060] The mice in the experimental group, gel group and control group were killed by cervical dislocation 21 days after the adrenal tissue was transplanted into the mouse spleen; the mice in the experimental group were killed by cervical dislocation 14 days, 28 days, 42 days and 56 days after the adrenal tissue was transplanted into the mouse spleen. After that, the mouse abdomen was fully wetted with 75% ethanol to disinfect the mouse, and the outer skin and endothelium were cut along the midline of the mouse abdomen with surgical scissors to fully expose the mouse abdominal cavity. After the intestinal tissue was pushed aside to expose the spleen, the spleen with transplanted adrenal tissue was removed with ophthalmic scissors, and then the removed spleen was placed in a pre-cooled PBS solution for washing 3-5 times. The washed spleen tissue was fixed and soaked in a 4% FPS solution for 2.5 hours to obtain the fixed spleen tissue, which was then transferred to 70% ethanol for short-term storage. The fixed spleen tissue was subjected to gradient dehydration, i.e., the fixed spleen tissue was placed in 75%, 80%, 85%, 90%, 95%, and 100% ethanol, respectively, and the immersion time for each concentration was 30 minutes to obtain the dehydrated spleen tissue. The dehydrated spleen tissue was paraffin embedded to make a paraffin specimen. The embedded spleen tissue was sliced ​​with a thickness of 5 mm to obtain paraffin sections. The prepared paraffin sections were subjected to HE and immunofluorescence staining to characterize the structure and function of the regenerated adrenal tissue in the spleen. The results of HE and immunofluorescence staining are shown in Figure 2, respectively. Figure 2 and Figure 3 .

[0061] like Figure 2 As shown in the figure, HE histological staining (from left to right: control group, gel group, experimental group) shows that the adrenal tissue structure of the experimental group after transplantation is intact, with clear tissue contours, and the internal structure of the adrenal tissue is relatively complete, which is obviously the same as the native adrenal structure. Figure 3 As shown in the figure, the immunofluorescence results of the adrenal tissues collected at different times in the experimental group showed that CYP21 can be normally expressed in the adrenal tissues transplanted into the spleen of mice, and the expression of CYP21 shows a certain upward trend as the transplantation time increases. The above results show that the transplanted adrenal tissue can normally play its own cortisol catalytic function.

[0062] Example 5: Regulation of host immunity by adrenal tissue after transplantation

[0063] Construction of an adrenal function-deficient mouse model: A certain number of mice were selected, and their adrenal tissues were surgically removed to construct an adrenal function-deficient mouse model.

[0064] The model mice were divided into an experimental group and an adrenalectomy group, and normal mice were used as a control group. The model mice in the experimental group were subjected to adrenal gel tissue transplantation according to the method described in Example 4, and the mice in the control group and adrenalectomy group were not treated in any way.

[0065] Blood was collected from the above groups of mice 4 weeks after the transplantation surgery, that is, the selected mice were immobilized. A lancet was used to puncture and collect blood at the intersection of the extended lines of the corners of the mouse's eyes and mouth. 500ul of mouse blood was collected using a blood collection tube containing an anticoagulant. Gently shake the collection tube containing the blood to allow the blood to fully contact the anticoagulant. A routine blood test was performed on the collected blood to obtain the number of lymphocytes in the blood. The number of lymphocytes in the blood of the mice in the control group, the mice in the experimental group after adrenal transplantation, and the mice in the adrenalectomy group after bilateral adrenal removal were compared. The results are shown in the table below. Figure 4 .

[0066] like Figure 4 As shown in the figure, by comparing the number of lymphocytes in the blood of mice transplanted with adrenal tissue gel and mice with bilateral adrenalectomy, it was found that the number of lymphocytes in the serum of mice transplanted with adrenal tissue gel 4 weeks later was significantly reduced by about 80% compared with that of mice with bilateral adrenalectomy. This result proves that transplanting adrenal tissue gel into the spleen can significantly alleviate the inflammatory response caused by adrenal deficiency or adrenal insufficiency.

[0067] Example 6: Regulation of host endocrine function by adrenal tissue after transplantation

[0068] The mice at 4, 6 and 8 weeks after the transplantation in Example 5 were used as the experimental group, the normal mice were used as the control group, and the bilaterally adrenalectomized mice were used as the adrenalectomy group.

[0069] Blood was collected from the mice in each group, that is, the selected mice were restrained. A lancet was used to puncture and collect blood at the intersection of the extended line of the mouse's eye corner and mouth corner. 500ul of blood was collected in a 1.5ml ep tube and allowed to stand at room temperature for 2h. The collected blood was then centrifuged at 3000rpm for 10min, and the light yellow liquid phase on the upper layer of the centrifuge tube was collected after centrifugation. The Elisa kit (EK8100-48, Lianke Bio) was used to detect the cortisol and ACTH content in the collected serum. The results are shown in Figure 5 .

[0070] like Figure 5As shown in the figure, by comparing the cortisol content and ATCH content in the serum of mice with adrenal transplantation and bilateral adrenalectomy, it was found that compared with bilateral adrenalectomy mice, the cortisol content in the serum of mice with adrenal transplantation 4 weeks later had a significant increase, and the cortisol concentration was 6010pg / ml at this time; at the same time, the ATCH content in the serum had a very obvious decrease, down to the lowest concentration of 967pg / ml. The above results show that the transplanted adrenal tissue can play its own endocrine function very well and can respond well to the host's own negative feedback regulation.

[0071] In summary, the treatment of adrenal tissue with the tissue gel provided by the present invention helps the transplanted adrenal tissue to quickly integrate with the host's vascular network, and can complete the transplantation while ensuring the structural integrity of the adrenal donor graft as much as possible, providing a basis for the rapid recovery of endocrine function of the transplanted tissue. By transplanting the adrenal tissue treated with the tissue gel of the present invention into the spleen of mice, the adrenal tissue can secrete normal and appropriate amounts of cortisol, so that the cortisol content in the serum is restored to a level close to the normal value, and the adrenal tissue can also be better integrated with the host, and can respond well to the regulation of the host's endocrine center. Based on the preparation scheme of the tissue gel of the present invention, the development of organ transplantation and regeneration schemes with high clinical relevance for the regeneration of different tissues is critical to the regeneration of endocrine organs in the spleen and the promotion of the transformation and application of adrenal tissue regeneration to clinical practice.

[0072] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A tissue gel, characterized in that: The tissue gel has adrenocorticotropic hormone and glucose dispersed in a hydrogel matrix material, and the mass ratio of the hydrogel matrix material, the adrenocorticotropic hormone and the glucose is (2.5-5): (3×10 -6 ~120×10 -6 ):

50.

2. The tissue gel according to claim 1, characterized in that The hydrogel matrix material is hyaluronic acid.

3. The method for preparing tissue gel according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: S1: dissolving glucose and ACTH in water to obtain ACTH solution; S2: adding the hydrogel matrix material to the ACTH solution, mixing and dissolving to obtain a tissue gel solution.

4. The preparation method according to claim 3, characterized in that: The concentration of the ACTH solution in step S1 is 3-120 ng / L; The concentration of glucose is 50 mg / mL.

5. Use of the tissue gel according to claim 1 or 2 in the preparation of a medicament for treating adrenal insufficiency.

6. A drug for treating adrenal insufficiency, characterized in that: include: The tissue gel according to claim 1 or 2, and adrenal tissue.

7. The drug according to claim 6, characterized in that The adrenal insufficiency is secondary adrenal insufficiency and / or primary adrenal insufficiency.

8. The drug according to claim 7, characterized in that The adrenal insufficiency includes congenital adrenal hyperplasia, unilateral or bilateral adrenalectomy, and iatrogenic Cushing's syndrome.

9. The method for preparing the drug according to any one of claims 6 to 8, characterized in that: The method comprises the following steps: i) cutting the adrenal tissue along the midline to obtain an adrenal tissue block; ii) premixing the adrenal tissue block with the tissue gel solution of claim 1 or 2, then taking it out and letting it stand to solidify, thereby obtaining the adrenal tissue gel.

10. The preparation method according to claim 9, characterized in that: The premixing conditions in step ii) are: temperature 37° C., time 10 to 15 min.

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