Use of rothia-derived outer vesicles for preparing an inducer of osteogenic differentiation of bone marrow mesenchymal stem cells

Abstract

The application provides application of a Roebel genus-derived outer vesicle in preparation of an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells, and relates to the technical field of biological medicine, wherein the Roebel genus-derived outer vesicle is obtained by using a super-speed centrifugation method, and experiments prove that the Roebel genus-derived outer vesicle has excellent effect of promoting osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and can significantly induce up-regulated expression of a marker protein of osteogenic differentiation, and can be used as an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells, and can be applied to preparation of anti-osteoporosis drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of extravesicles derived from *Rhodotorula spp.* in the preparation of an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells. Background Technology

[0002] Osteoporosis (OP) is a systemic metabolic disease characterized by increased bone fragility and a higher probability of fractures. Microscopically, it manifests as a reduction in bone trabeculae and destruction of bone microstructure. Currently, bone metabolism imbalance is considered the primary cause of this disease. Osteoporosis can occur in both sexes and at any age, but it is most common in postmenopausal women and the elderly. Currently, there are no effective interventions or preventative measures for this disease in clinical practice.

[0003] Extracellular vesicles are lipid membrane vesicles of varying sizes secreted by cells outside the cell. These vesicles can transport various molecules from the producing cell to the target cell. The three main subtypes of extracellular vesicles are microvesicles, exosomes, and apoptotic bodies. Among the different types of extracellular vesicles, exosomes are nanoscale vesicles (30 nm to 150 nm) found in various organisms. Increasing evidence suggests that extracellular vesicles play a crucial role in intercellular communication, influencing physiological and pathological processes, and have been extensively studied as nanotherapeutic agents, drug delivery carriers, and biomarkers. Currently, research mainly focuses on extracellular vesicles derived from mammals and plants, while reports on bacterial extracellular vesicles and their composition and function are rare.

[0004] Roseburiae, a genus of bacteria, has received widespread attention in recent years. It is a commensal bacterium belonging to the class Clostridium, order Clostridium, family Trichophyceae, genus Roseburiae. It is a Gram-positive anaerobic bacterium, currently comprising five species (Roseburia intestinalis, R. hominis, R. inulinivorans, R. faecis, and R. cecicola), accounting for 2-31% of the total healthy gut bacteria. It produces short-chain fatty acids, particularly butyrate, which affects colonic motility. There are reports that Roseburiae can improve gut biodiversity and enhance glucose tolerance. Dysregulation (insufficiency) of Roseburiae may affect multiple metabolic pathways, participating in conditions including irritable bowel syndrome, obesity, type 2 diabetes, and neurological disorders. However, no research has yet reported on the application of Roseburiae-derived exovesicles in the treatment of osteoporosis. Summary of the Invention

[0005] The technical problem solved by this invention is to provide the application of exovesicles derived from *Rhodotorula spp.* in the preparation of anti-osteoporosis drugs.

[0006] To address the aforementioned technical problems, this invention provides the application of extravesicles derived from *Rhodotorula spp.* in the preparation of an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells.

[0007] Optionally, the method for preparing the extravesicles derived from *Rochetomyces* includes:

[0008] Step S1: After subculturing and activating the genus *Rochetomyces*, add it to the culture medium, culture it under closed conditions with shaking, perform the first centrifugation, extract the supernatant, filter it using the first bacterial sieve, and obtain the first supernatant.

[0009] Step S2: Centrifuge the first supernatant a second time to extract the supernatant, and filter it using a second bacterial sieve to obtain the second supernatant;

[0010] Step S3: Centrifuge the second supernatant for the third time to extract the supernatant, and filter it using a third bacterial sieve to obtain the third supernatant;

[0011] Step S4: Centrifuge the third supernatant for the fourth time to extract the supernatant and obtain the fourth supernatant;

[0012] Step S5: Centrifuge the fourth supernatant for the fifth time to remove the supernatant. The resulting precipitate is the exovesicle from the genus Rocherton.

[0013] Optionally, the genus *Roseburia* is *Roseburia intestinalis*.

[0014] Optionally, in step S1, the rotation speed of the first centrifugation treatment is 1500g to 2000g, and the time is 20min to 30min.

[0015] Optionally, in step S2, the rotation speed of the second centrifugation treatment is 9500g to 10500g, and the time is 20min to 30min.

[0016] Optionally, in step S3, the rotation speed of the third centrifugation process is 9500g to 10500g, and the time is 30min to 40min.

[0017] Optionally, in step S4, the rotation speed of the fourth centrifugation treatment is 9500g to 10500g, and the time is 30min to 40min.

[0018] Optionally, in step S5, the rotation speed of the fifth centrifugation process is 145,000 g to 155,000 g, and the time is 90 min to 120 min.

[0019] Optionally, the pore size of the second bacterial sieve is 0.45 μm.

[0020] Optionally, the pore size of the third bacterial sieve is 0.22 μm.

[0021] Compared with related technologies, this invention uses ultracentrifugation to obtain external vesicles from *Rhodotorula spp.* and demonstrates through experiments that external vesicles from *Rhodotorula spp.* have excellent effects in promoting osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and can significantly induce the upregulation of osteogenic differentiation marker proteins. They can be used as osteogenic differentiation inducers of bone marrow mesenchymal stem cells and can be applied to the preparation of anti-osteoporosis drugs. Attached Figure Description

[0022] Figure 1 The image shown is a transmission electron microscope image of an external vesicle derived from the genus *Rosella* in Example 1 of this invention.

[0023] Figure 2 This is a particle size distribution diagram of the extravesicles derived from *Rochetomyces* in Example 1 of the present invention;

[0024] Figure 3 The figure shows the effect of different concentrations of extravesicular solutions derived from *Rhodotorula spp.* on the activity of hBMSCs cells in Example 3.

[0025] Figure 4 Light micrographs of alkaline phosphatase staining in osteogenic differentiation of hBMSCs induced by different concentrations of extravesicular solutions derived from Rochetomyces in Example 4.

[0026] Figure 5 Alizarin Red stained light micrographs of hBMSCs osteogenic differentiation induced by different concentrations of extravesicular solutions derived from Rochetomyces in Example 4.

[0027] Figure 6 This is a gel electrophoresis image of marker proteins for osteogenic differentiation of hBMSCs induced by different concentrations of extravesicular solutions derived from *Rhodotorula spp.* in Example 5. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0029] It should be noted that, unless otherwise specified, the features in the embodiments of this invention can be combined with each other. The terms "comprising," "including," "containing," and "having" are non-limiting, meaning that other steps and other components that do not affect the results can be added. The above terms cover the terms "composed of" and "substantially composed of." Unless otherwise specified, the materials, equipment, and reagents are commercially available.

[0030] This invention provides the application of external vesicles derived from *Rhodotorula spp.* in the preparation of an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells.

[0031] In this invention, extravesicles derived from *Rhodotorula spp.* were obtained using ultracentrifugation. Experiments have demonstrated that these extravesicles have excellent effects in promoting osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and can significantly induce the upregulation of osteogenic differentiation marker proteins. They can be used as osteogenic differentiation inducers of bone marrow mesenchymal stem cells and can be applied to the preparation of anti-osteoporosis drugs.

[0032] In some embodiments of the present invention, the method for preparing the exovesicles derived from *Rochetomyces* includes:

[0033] Step S1: After subculturing and activating the genus *Rochetomyces*, add it to the culture medium, culture it under closed conditions with shaking, perform the first centrifugation, extract the supernatant, filter it using the first bacterial sieve, and obtain the first supernatant.

[0034] Step S2: Centrifuge the first supernatant a second time to extract the supernatant, and filter it using a second bacterial sieve to obtain the second supernatant;

[0035] Step S3: Centrifuge the second supernatant for the third time to extract the supernatant, and filter it using a third bacterial sieve to obtain the third supernatant;

[0036] Step S4: Centrifuge the third supernatant for the fourth time to extract the supernatant and obtain the fourth supernatant;

[0037] Step S5: Centrifuge the fourth supernatant for the fifth time to remove the supernatant. The resulting precipitate is the exovesicle from the genus Rocherton.

[0038] In some embodiments of the present invention, the genus *Roseburia* is, by way of example, *Roseburia intestinalis*.

[0039] In some embodiments of the present invention, in step S1, the rotation speed of the first centrifugation treatment is 1500g to 2000g, and the time is 20min to 30min; the pore size of the first bacterial sieve is 0.45μm.

[0040] In some embodiments of the present invention, in step S2, the rotation speed of the second centrifugation treatment is 9500g to 10500g, the time is 20min to 30min, and the pore size of the second bacterial sieve is 0.45μm.

[0041] In some embodiments of the present invention, in step S3, the rotation speed of the third centrifugation treatment is 9500g to 10500g, and the time is 30min to 40min; the pore size of the third bacterial sieve is 0.22μm.

[0042] In some embodiments of the present invention, in step S4, the rotation speed of the fourth centrifugation process is 9500g to 10500g, and the time is 30min to 40min.

[0043] In some embodiments of the present invention, in step S5, the rotation speed of the fifth centrifugation process is 145,000 g to 155,000 g, and the time is 90 min to 120 min.

[0044] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The human bone marrow mesenchymal stem cells (hBMSCs) used in the embodiments of the present invention were purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd., and the product name is OriCell. ® Adult bone marrow mesenchymal stem cells, model number HUXMA-01001. The *Roseburia intestinalis* strain used was purchased from Ningbo Taisite Biotechnology Co., Ltd., product name *Roseburia intestinalis*-DSM 14610, catalog number TS253874. This invention... Figure 3 In this context, ns indicates that there is no significant difference.

[0045] Example 1: Extraction of exovesicles from *Rhodotorula spp.*

[0046] 1.1. After subculturing and activating *Roseburia intestinalis*, the bacteria are added to the culture medium and cultured under closed conditions with shaking. A first centrifugation is then performed to extract the supernatant, which is then filtered using a first bacterial sieve to obtain the first supernatant. The first centrifugation is carried out at 2000g for 25 minutes. The pore size of the first bacterial sieve is 0.45μm. The culture medium comprises: 10 parts tryptone, 10 parts beef extract, 3 parts yeast extract, 5 parts glucose, 5 parts sodium chloride, 1 part soluble starch, 0.5 parts L-cysteine ​​hydrochloride, 3 parts sodium acetate, 0.001 parts resazurin, and 1000 parts deionized water.

[0047] 1.2 The first supernatant is centrifuged a second time to extract the supernatant, and then filtered using a second bacterial sieve to obtain a second supernatant; wherein, the second centrifugation is performed at a speed of 10000g for 25min; and the pore size of the second bacterial sieve is 0.45μm;

[0048] 1.3. The second supernatant is centrifuged a third time to extract the supernatant, and then filtered using a third bacterial sieve to obtain the third supernatant; the third centrifugation is performed at a speed of 10000g for 35min; the pore size of the third bacterial sieve is 0.22μm.

[0049] 1.4. The third supernatant is centrifuged for a fourth time to extract the supernatant and obtain a fourth supernatant; the fourth centrifugation is performed at a speed of 10000g for 35min.

[0050] 1.5. The fourth supernatant is centrifuged a fifth time to remove the supernatant. The resulting precipitate is the exovesicle from *Rochetomyces*. The fifth centrifugation is performed at 150,000 g for 105 min.

[0051] Transmission electron microscopy was used to identify the obtained extravesicles derived from the genus *Rochetomyces*. Figure 1 Transmission electron microscopy images of external vesicles derived from the genus *Rochetomyces*. Figure 1 It can be seen that the morphology of the external vesicles derived from *Rochetomyces* is a typical membranous "cup-and-disc" structure. The particle size of the external vesicles from *Rochetomyces* was determined using a nanoparticle tracking analyzer. Figure 2 This is a particle size distribution diagram of exovesicles derived from the genus *Rochetomyces*. From... Figure 2 It can be seen that the outer vesicles from the genus *Rochetomyces* have a particle size concentrated around 110 nm.

[0052] Example 2: Determination of the concentration of extravesicular proteins from *Rhodotorula spp.*

[0053] The obtained extravesicles from *Rochetomyces* were tested using a BCA protein assay kit. The specific steps are as follows:

[0054] (1) Dilute the protein standards with sterile PBS buffer to prepare protein standards with concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg / mL; add 20 μL of each concentration of protein standard to the standard wells of a 96-well plate to prepare a standard curve.

[0055] (2) Dissolve the extravesicles of *Rhodotorula spp.* obtained in Example 1 in sterile PBS buffer to obtain an extravesicle solution of *Rhodotorula spp.*; take 1 μL of the extravesicle solution of *Rhodotorula spp.* and dilute it to 20 μL with PBS buffer, and add it to the sample wells of a 96-well plate;

[0056] (3) Add 200 μL of BCA working solution to the standard wells and sample wells of the 96-well plate respectively, and place at 37°C for 30 min;

[0057] (4) Measure the A562 value using an enzyme-linked immunosorbent assay (ELISA) reader;

[0058] (5) Calculate the protein concentration of the extravesicular vesicle solution from Rochetomyces based on the standard curve.

[0059] The protein concentration in the extravesicular vesicle solution derived from *Rhodotorula fragilis* obtained in Example 2 was determined to be 1 mg / mL.

[0060] Example 3: Effect of Rhodotorula spp.-derived external vesicles on the activity of human bone marrow mesenchymal stem cells (hBMSCs)

[0061] The details are as follows:

[0062] (1) The extravesicles of *Rhodotorula spp.* obtained in Example 1 were added to α-MEM complete medium to obtain a first extravesicle solution and a second extravesicle solution, wherein the concentration of *Rhodotorula spp.* extravesicles in the first extravesicle solution was 1 μg / ml and the concentration of *Rhodotorula spp.* extravesicles in the second extravesicle solution was 10 μg / ml; α-MEM complete medium was used as a blank control solution.

[0063] (2) P5 generation human bone marrow mesenchymal stem cells (hBMSCs) were seeded and cultured in 96-well plates at a seeding density of 5 × 10⁶. 3 / well; after the cells adhered, they were incubated for 72h with the first outer vesicle solution, the second outer vesicle solution, and the blank control solution obtained in step (1); CCK-8 reagent was added to each well at a ratio of 10:1, and the cells were incubated at 37℃ for 2h. The absorbance at 450nm was measured using a UV spectrophotometer. The results are shown in the figure. Figure 3 .

[0064] Figure 3 This figure shows the effect of different concentrations of *Rhodotorula spp.* external vesicle solutions on the viability of hBMSCs. It should be noted that... Figure 3 The concentrations of 0 μg / ml, 1 μg / ml, and 10 μg / ml correspond to the experimental results of the blank control solution, the first outer vesicle solution, and the second outer vesicle solution, respectively. From... Figure 3 It can be seen that after incubation with the external vesicles derived from Rochetomyces for 72 hours, the proliferation activity of human bone marrow mesenchymal stem cells (hBMSCs) was not significantly affected, indicating that the external vesicles derived from Rochetomyces of the present invention are not toxic to human bone marrow mesenchymal stem cells (hBMSCs).

[0065] Example 4: Experiment on the promotion of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) by external vesicles derived from Rochetomyces.

[0066] The details are as follows:

[0067] (1) The osteogenic medium was α-MEN complete medium containing dexamethasone, ascorbic acid and β-glycerophosphate; wherein the concentration of dexamethasone was 100 nmol / L, the concentration of ascorbic acid was 50 nmol / L and the concentration of β-glycerophosphate was 10 nmol / L; the extravesicles of *Rhodotorula spp.* obtained in Example 1 were added to the osteogenic medium to obtain the first *Rhodotorula spp.* extravesicle solution and the second *Rhodotorula spp.* extravesicle solution, wherein the concentration of *Rhodotorula spp.* extravesicles in the first *Rhodotorula spp.* extravesicle solution was 1 μg / ml and the concentration of *Rhodotorula spp.* extravesicles in the second *Rhodotorula spp.* extravesicle solution was 10 μg / ml; the osteogenic medium was used as a blank control solution.

[0068] (2) P5 generation human bone marrow mesenchymal stem cells (hBMSCs) were seeded and cultured in 12-well plates at a seeding density of 5 × 10⁶. 5 / well; After the cells adhered, they were cultured using the first Rhodotorula spp.-derived extravesicular vesicle solution, the second Rhodotorula spp.-derived extravesicular vesicle solution, and the blank control solution obtained in step (1) as culture media, and the culture medium was changed every 3 days. After 3 days of osteogenic differentiation culture, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 15 min. Then, alkaline phosphatase staining solution was added to each well for staining. The cells were incubated at room temperature for 60 min, and the non-specifically bound alkaline phosphatase staining solution was washed away with PBS buffer. The stained images are shown in the figure. Figure 4 .

[0069] (3) P5 generation human bone marrow mesenchymal stem cells (hBMSCs) were seeded and cultured in 12-well plates at a seeding density of 5 × 10⁶. 5 / well; After the cells adhered, they were cultured using the first Rhodotorula spp.-derived extravesicular vesicle solution, the second Rhodotorula spp.-derived extravesicular vesicle solution, and the blank control solution obtained in step (1) as culture media. The culture medium was changed every 3 days. After 10 days of osteogenic differentiation culture, the culture medium was removed, and the cells were fixed with 75% ethanol for 15 min. Then, Alizarin Red S staining solution was added to each well for staining. The cells were incubated at room temperature for 20 min. Non-specifically bound Alizarin Red S staining solution was removed using PBS buffer. The stained images are shown in the figure. Figure 5 .

[0070] Figure 4 Light micrographs showing alkaline phosphatase staining in hBMSCs induced by different concentrations of external vesicle solutions derived from *Rhodotorula spp.* Figure 5 Alizarin red stained light micrographs of hBMSCs induced by different concentrations of external vesicle solutions derived from Rochetomyces spp. Figure 4 and Figure 5The values ​​of 0 μg / ml, 1 μg / ml, and 10 μg / ml correspond to the experimental results of the blank control solution, the exovesicle solution derived from *Rhizoctonia solani* var. *sinensis*, and the exovesicle solution derived from *Rhizoctonia solani* var. *sinensis*, respectively. From... Figure 4 and Figure 5 It can be seen that the staining of cells treated with blank control solution, cells treated with solution of external vesicles from the first genus of Rochetomyces, and cells treated with solution of external vesicles from the second genus of Rochetomyces increased in intensity in that order. This indicates that external vesicles from Rochetomyces can significantly promote osteogenic differentiation of human bone marrow mesenchymal stem cells, and that human bone marrow mesenchymal stem cells have successfully differentiated into osteoblasts.

[0071] Example 5: Effect of extravesicles derived from *Rhodotorula fragilis* on osteogenic marker proteins in human bone marrow mesenchymal stem cells (hBMSCs)

[0072] The induction effect was detected by Western blot analysis, as detailed below:

[0073] (1) The extravesicles of *Rhodotorula spp.* obtained in Example 1 were added to α-MEM complete medium to obtain a third *Rhodotorula spp.* extravesicle solution and a fourth *Rhodotorula spp.* extravesicle solution, wherein the concentration of *Rhodotorula spp.* extravesicles in the third *Rhodotorula spp.* extravesicle solution was 1 μg / ml and the concentration of *Rhodotorula spp.* extravesicles in the fourth *Rhodotorula spp.* extravesicle solution was 10 μg / ml; α-MEM complete medium was used as a blank control solution.

[0074] (2) P5 generation human bone marrow mesenchymal stem cells (hBMSCs) were seeded in 60 mm cell culture dishes at a seeding density of 5 × 10⁻⁶. 5 / well; after the cell density grows to 80%, the cells are cultured in three solutions obtained in step (1) (exovesicle solution from the third genus *Rosnezoffii*, exovesicle solution from the fourth genus *Rosnezoffii*, and blank control solution), respectively. The cells are lysed in RIPA lysis buffer for 30 min, centrifuged at 12000g for 10 min, and the total protein concentration of the supernatant is determined using a BCA protein assay kit. After balancing with protein loading buffer, the cells are boiled in 100℃ water for 10 min. 15g of protein is extracted from each group corresponding to the above three solutions and added to a 10% SDS-PAGE gel. 60 Separate the proteins by constant voltage electrophoresis for 2 hours. Transfer the separated proteins from each group to a PVDF membrane. Divide each PVDF membrane into four test membranes. Block the test membranes in 5% skim milk for 1 hour. Then, incubate the four test membranes from each group overnight at 4°C with specific primary antibodies such as RUNX2 (1:1000), ALP (1:1000), OPN (1:1000), and β-actin (1:1000). After recovering the primary antibodies, wash the membranes with TBST for 30 minutes. Incubate the membranes with goat anti-rabbit secondary antibody (1:5000) at room temperature for 1 hour, followed by washing with TBST for another 1 hour. Apply rapid chemiluminescence buffer evenly to the membranes, let stand for 2 minutes, then remove the membranes and place them in a chemiluminescence imaging system for detection. Results are shown in [Figure number missing]. Figure 6 .

[0075] Figure 6 Gel electrophoresis images of marker proteins for osteogenic differentiation of hBMSCs induced by different concentrations of extravesicular solutions derived from *Rhodotorula spp.* Figure 6 The values ​​of 0 μg / ml, 1 μg / ml, and 10 μg / ml correspond to the experimental results of the blank control solution, the exovesicle solution derived from *Rhizobium spp.* (third), and the exovesicle solution derived from *Rhizobium spp.* (fourth). Figure 6 It can be seen that the expression of osteogenic marker proteins such as ALP, RUNX2, and OPN is significantly increased, indicating that the external vesicles derived from Rochetomyces can significantly induce the upregulation of osteogenic differentiation marker proteins, significantly promote osteogenic differentiation of human bone marrow mesenchymal stem cells in a dose-dependent manner.

[0076] Furthermore, it should be noted that although the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. Enterococcus faecalis ( Roseburia intestinalis Application of DSM 14610-derived external vesicles in the preparation of an inducer for osteogenic differentiation of bone marrow mesenchymal stem cells.

2. The application according to claim 1, characterized in that, The Enterococcus ( Roseburia intestinalis Methods for preparing exovesicles derived from DSM 14610 include: Step S1, add Enterococcus faecalis ( Roseburia intestinalis After DSM 14610 was activated by subculture, it was added to the culture medium and cultured under closed conditions with shaking. After the first centrifugation, the supernatant was extracted and filtered using a first bacterial sieve to obtain the first supernatant. The first centrifugation was performed at a speed of 1500g to 2000g for 20min to 30min. Step S2: The first supernatant is centrifuged a second time to extract the supernatant, and then filtered using a second bacterial sieve to obtain a second supernatant; the second centrifugation is performed at a speed of 9500g to 10500g for 20min to 30min; the pore size of the second bacterial sieve is 0.45μm. Step S3: The second supernatant is centrifuged for a third time to extract the supernatant, and then filtered using a third bacterial sieve to obtain the third supernatant; the third centrifugation is performed at a speed of 9500g to 10500g for 30min to 40min; the pore size of the third bacterial sieve is 0.22μm. Step S4: The third supernatant is centrifuged for a fourth time to extract the supernatant and obtain the fourth supernatant; the speed of the fourth centrifugation is 9500g to 10500g and the time is 30min to 40min. Step S5: Centrifuge the fourth supernatant a fifth time to remove the supernatant; the resulting precipitate is Enterococcus faecalis (C. faecalis). Roseburia intestinalis The fifth centrifugation was performed on exovesicles derived from DSM 14610. The centrifugation was performed at a speed of 145,000 g to 155,000 g for 90 min to 120 min.

Images