WO2025032360A1

WO2025032360A1 - The structure obtained from the bioreactor with fixed bed hydrogel for wound healing

Info

Publication number: WO2025032360A1
Application number: PCT/IB2023/058036
Authority: WO
Inventors: Elahe ABEDINI
Original assignee: Individual
Current assignee: Individual
Priority date: 2023-08-09
Filing date: 2023-08-09
Publication date: 2025-02-13
Anticipated expiration: 2026-02-09

Abstract

The bioreactor with fixed bed hydrogel for wound healing, which the main feature of this bioreactor is the constant exchange of the culture medium throughout the system, improving the mass transfer of nutrients and waste materials, as well as applying minimum shear stress to the cells. The cell culture bioreactor is a dynamic and continuous system that has the ability to control the entry and exit of the culture medium liquid, install oxygen, carbon dioxide, pressure, temperature and dissolved oxygen sensors. The outer shell material can be changed from USP class VI hard plastic or glass to stainless steel. There are different dimensions and shapes to conduct studies in tissue engineering and stem cells in this bioreactor.

Description

The structure obtained from the bioreactor with fixed bed hydrogel for wound healing

C12N 5/00

Hydrogel-containing medical articles and methods of using and making the same

United States Patent Application 20050214376

Medical articles including a hydrophilic water-swellable hydrogel and methods of using and making the articles are provided. The hydrogel may include a cross-linked mixture of a biocompatible polymer and a protein, such as polyethylene glycol and a soy protein. The hydrogel may further include an agent, such as diazolidinyl urea and iodopropynyl butylcarbamate, dispersed within the hydrophilic water-swellable hydrogel.

Methods and compositions for the treatment of wounds

United States Patent 6299898

Devices and methods for enhancing the healing of wounds, especially chronic wounds (e.g., diabetic wounds), involving the use of keratinocytes are described. Keratinocytes are grown on a transplantable solid support (e.g., collagen-coated beads), and the keratinocyte-coated solid support is placed in an enclosure. The enclosure, in turn, is placed in the wound for use as an interactive wound healing promoter.

WOUND HEALING FORMULATION

United States Patent Application 20180021385

The present disclosure relates to a method for producing a composition, wherein the method comprises the steps of co-culturing immortalized fibroblasts and immortalized keratinocytes, thereby producing secretion; separating the secretion from the fibroblasts and keratinocytes; and providing a pharmaceutically acceptable composition comprising the secretion. The present disclosure also relates to the composition obtainable by the method, wherein the composition preferably is a pharmaceutical composition for medical use, preferably for use in the treatment of a wound, preferably a chronic or acute wound.

System for the treatment of wounds

United States Patent 6197330

Bioreactors, systems, and methods for vascularizing tissue constructs

United States Patent 9226494

A modular bioreactor is provided that includes an upper biochamber; a lower biochamber; and an intermediate biochamber that is positioned between the upper biochamber and the lower biochamber. Each biochamber of the bioreactor is in fluid communication with each other biochamber of the bioreactor and includes an interior wall, which defines a centrally-disposed cavity for each biochamber, and an inflow port and an outflow port that are in fluid communication with each centrally disposed cavity. Systems and methods for vascularizing a tissue construct are also provided.

Systems to promote healing at a site of a medical device

United States Patent 10576185

Disclosed herein are systems for promoting healing of a wound or surgical incision at a medical device site (e.g., implanted medical device) in a subject, by administering a microporous gel to the medical implant site. Also disclosed are systems for the treatment and prevention of infection at a medical implant site in a subject, by administering a microporous gel to the medical implant site. The microporous gel may be fluidic during application and annealed or cross-linked after application. The microporous gels may contain various therapeutic agents, including antibiotics and analgesics, throughout the gel.

The aforementioned cell culture system does not have an internal sample, and similar external samples also have synthetic scaffolds that cannot be injected into body tissues and organs, and their cost is 6-8 times higher than this system.

Because trauma is the leading cause of death in the country, the society faces a variety of injuries and damages resulting from accidents. One side of this spectrum is the physical problems and different injuries that happen to people who are hurt in accidents and traumas. Actually, trauma occurs when the skin and tissues underneath get hurt and damaged, often causing wounds formation.

The main goal of treating wounds is to make them heal as quickly as possible. This means preventing infections and complications, as well as minimizing any negative effects on how the wound looks and functions once it has healed. The current ways of healing wounds are not enough and have some problems, so it is important to find a new treatment approach. The strong ability of cell-loaded scaffolds to help tissue grow back has encouraged us to use this method to treat wounds caused by injuries. In this project, we will create a special container called a bioreactor and use it to grow cells from fat. We will then place these cells into a gel-like material that can be injected into wounds to help them heal. This will allow us to create a device that can treat many different types of injuries.

Since the end of the 19th century, the Petri dish has been used as the most reliable device for cell biology experiments. In fact, standard Petri dish cultures are still used worldwide. The goals of using these containers include the direct attachment and adhesion of grown cells to synthetic surfaces such as tissue culture plastic, coating these surfaces with extracellular proteins such as laminin, collagen or nutritional cells such as mouse embryonic fibroblast (MEF) for attachment cells and finally the creation of a culture medium pool containing signaling and nutritional molecules suitable for cell growth. In Petri dish, cells necessarily grow in two dimensions and in dense colonies with distinct borders.

In Petri dish, cells necessarily grow in two dimensions and in dense colonies with distinct borders. These cells expand in size and number and merge with other colonies. This way of growth and crossing limits the growth and proliferation of more cells in the culture medium. In practice, Petri dish culture lacks cell-cell or cell-matrix interaction, lacks cell organization and signaling, and does not have the ability to communicate with other systems. Therefore, the results obtained from Petri dish culture are not always predictable for tissues and organs (in vivo) and are problematic to convert to the in vivo situation in preclinical and clinical studies. In contrast, controllable cell culture systems or animal models allow access to all the developmental capabilities of stem cells and are reliable for evolutionary studies, disease pathogenesis, and drug toxicity testing. However, animal models have limitations, such as the fact that in disease models or toxic pathophysiology studies, they are very little close to human conditions, and the obtained results are not reliable due to different physiology and interspecies differences. On the other hand, in animal models, there is less control over the cellular environment and moment-to-moment monitoring of the results is considered the main challenge, but compared to them, in vitro systems are better and easier to control and evaluate, and the obtained results are reliable. Therefore, technologies based on biological reactors can make it possible to predict the results of in vivo studies in laboratory conditions (in vitro).

Our awareness and knowledge of stem cell biology has greatly increased through the development of new culture media and the use of biomechanics and bioreactors, so that these devices and equipment produce specific parts of specific tissues. The importance of inductive signals in the 3D culture environment has been known for several years and simple culture environments (conditioning media) obtained from the culture of embryonic and mesenchymal stem cells have been used for cell differentiation. Despite this, this type of cell culture has limitations such as small size (less than 0.5 mm), lack of control over cell population and signals, and absence of extracellular matrix. For this reason, since the 1990s, the main goal of using three-dimensional tissue structures was to increase the ability to build and produce culture media using scaffolding materials that temporarily provide a biocompatibility model for tissue development and growth. After them, bioreactor systems have provided environmental control and biophysical and biochemical signaling. This achievement was named as biomimetic paradigm, which gave rise to the idea and thought that the combination of scaffolds with bioreactors can govern environmental and morphological events in vivo in vitro. During the development of the original tissue, cells are exposed to a myriad of signals, including contact with various types of cells, extracellular matrix, cytokines, growth factors, and all natural dynamic factors, and it was actually based on the quasi-biological paradigm that in vitro systems using Biomaterial scaffolds were designed to show all the characteristics of cell niches, supporting factors for matrix construction, cell growth and proliferation, and finally tissue formation. The advances made in the field of biomaterials made it possible to build three-dimensional scaffolds with specific structural and molecular aspects (such as shape, permeability, pore size, surface roughness), mechanical properties, and defined levels of construction and destruction, and this diversity in materials caused various geometric forms of scaffolds have been provided in the form of fibers, meshes, plates, sponges and hydrogels.

In order to induce cell growth in three-dimensional sections and to support tissue development, it is very important to provide mass transfer of materials (biomass) into and out of all cells, which is possible by using dynamic culture systems such as bioreactors. In static environments such as Petri dishes or flasks, mass transfer is done based on diffusion and is generally limited to 0.2 mm with the increase in tissue development due to the decrease in oxygen diffusion and the increase in the concentration of harmful metabolites. In bioreactors, mobility, diffusion, and loading (active addition of the required factor) causes sequential and continuous transfer, allowing growth and development of tissues on the millimeter to centimeter scale.

By determining the direction and amount of medium flow, electrical stimulation and loading, bioreactors cause movement or diffusion of the medium, allow online control and monitoring of temperature, pH, oxygen, nutrient concentration of the medium and provide the needs of the studied tissue.

Injectable gel-shaped scaffolds are suitable for use in various tissue engineering methods such as drug transfer and delivery, treatment of damaged tissues and their replacement. These hydrogels are promising agents in therapeutic products due to their low invasiveness and easy ability to fill tissue defects.

Commonly, various synthetic hydrogels are used in tissue engineering, which can be converted into the desired structure due to their special mechanical and chemical properties, however, it has recently been reported that injectable hydrogels derived from the extracellular matrix have advantages such as bio Compatibility and immunoreactivity are low compared to synthetic hydrogels.

The extracellular matrix has a high potential in the differentiation of stem cells towards target cells. For this reason, the use of acellular tissue can be used as a natural scaffold in tissue engineering. Also, acellular tissues stimulate the immune system less, and for this reason, the success rate in tissue grafting using acellular matrix is high. Despite the many advantages of using natural matrices in tissue engineering, the methods used in tissue acellularization can seriously overshadow its applicability.

Currently, there are many studies on acellularization of various tissues, such as dermis, liver, bladder, and small intestine. ECM of the small intestine in the dry state contains 90% collagen, which is mostly collagen type I and a small amount of collagen type III, IV, V and VII, and also contains adhesion molecules such as fibronectin, laminin, entactin, glycosaminoglycans, and in addition contains some growth factors such as bFGF, VEGF, TGFβ, that maintaining these huge growth and differentiation stimulating sources depends a lot on the acellularization method that can maintain these compounds as much as possible (11). A lot of research has been done on ECM of the small intestine and its regenerative effect in the world, and in some cases it has even gone as far as pre-clinical tests and has been very successful, and even led to the production of the commercial product OASIS, which is processed from the small intestine of pigs. For example, in a study conducted on acellular pig small intestine by Jian Zhang and his colleagues in 2011, it has been shown that the small intestine has good mechanical properties, high water exchange and angiogenesis, and therefore has a lot of regenerative power in repairing the abdominal wall.

Based on the tissue engineering paradigm, tissue production consists of several phases, starting with sampling (phase 1) and isolation of cells (phase 2), increasing the number of isolated cells in vitro (phase 3), and tissue production (phase 4) and its three-dimensional maturation continued (phase 5) and subsequently ends with the use of the structure in test systems or transplantation (phase 6).

In these stages, there are different prospects for using biological reactor systems in order to improve the efficiency and guarantee of each of these stages. The direct impact of these systems in the process of tissue engineering including cell proliferation and expansion in vitro (phase 3) and better maturation of them (phase 5). In phase 3, the reactors reduce the cost and improve the efficiency of the process by reducing the possibility of contamination (13). With the beginning of the tissue production stage (phase 4), the conditions of the culture environment change from two-dimensional to three-dimensional. Like in vivo evolution, in vitro tissue evolution relies on spatial arrangement and temporal organization, which is caused by the synthesis, mass transfer, binding and degradation of molecules. Providing effective materials for a complex three-dimensional structure is the main challenge in tissue engineering and is a limiting factor for the production and development of large engineering tissues and their grafting.

Bioreactors are used therapeutically for the proliferation and expansion of various types of cells. There are many limitations in the clinical application of the inherent cells of a tissue, embryonic stem cells, mature and induced multipotent stem cells, which is due to the deficiency in the methods of reproduction and expansion of these cells in laboratory conditions. According to studies, the culture and proliferation of hematopoietic stem cells with common methods in T flasks is associated with a high risk of contamination, therefore, the proliferation of these cells in laboratory conditions by using a microchannel perfusion bioreactor in combination with hallow-fiber reactors and the spinner-flask has been improved.

After the transplantation of mesenchymal stem cells, the issue of transplant rejection in the host is raised. This effect can be caused by stimulating the host's immune system. For this reason, adjusting the immune system can be helpful in this field. The study showed that in order to achieve the clinical application of these cells, cell proliferation can be continued using a rotating bioreactor under low-stress conditions until these cells find the ability to differentiate into adipogenesis, osteogenesis, and chondrogenesis cell lines.

The ability of embryonic stem cells to self-renew or differentiate into various cell lines has created a desire to use these cells in cell therapy, tissue engineering, and model systems for drug screening. For this purpose, the main problem is to create suitable conditions for the proliferation of these cells without using additional nutrient layers and medium. In this field, significant progress has been made regarding the growth and proliferation of rodent embryonic stem cells using stirred vessels bioreactors with microcarriers (21, 22). In micro carrier-base reactors, the number of cells can increase 34 to 45 times in 8 hours (23). In a study conducted by Ahsan and Nerem, embryonic stem cells in the fluid based shear stress reactor increased the proliferation and expression of proteins such as endothelial markers fetal liver kinase 1, VE-cadherin and platelet endothelial cell adherence molecule (PECAM) can have the ability to transform into a pseudo-vein structure.

Rotatoring bioreactors are a system that has the ability to exchange and transport oxygen in order to induce chondrogenesis. In this structure, the loaded cells (seeded) inside the meshes or polymer sponge with large pores are suspended in the flow path of the medium between two concentric cylinders.

Perfusion bioreactor is used for the culture and engineering of heart tissue, in which the medium is injected into canalized elastomer scaffolds.

Several studies show that SIS (Small intestinal submucosa) scaffold creates useful structures in body tissues such as bone, skin, vocal cords, esophageal tissue, bile ducts, tunica albuginea, kidney, ureter, and abdominal wall. Many studies have been conducted on the effectiveness of SIS, but the study of SIS-based injectable hydrogels in vivo is rare, so the development and evolution of this structure will be valuable in medical biomaterials research. The centrality of this bioreactor includes the use of dense fixed bed with customized micro carriers, which will use exclusive hydrogel scaffold in this design. This matrix contains a variety of cell adhesion molecules and creates a dense and large growth surface with cell penetration pores. The simplicity of the process that continues from the injection of cells into the biomass to the production of the final product forms the central core and innovative part of this platform. Therefore, the cells are immobile and fixed inside the fixed bed and the system works as a perfusion model without the need to centrifuge the cultured cells.

Among the goals of the above invention, the following can be mentioned: the production of a laboratory model biological reactor and the investigation of its synergistic effect between cells and the culture medium in the form of mass transfer to support tissue development, the production of the maximum number of fat-derived stem cells on an injectable gel scaffold with and without cell differentiation, creating a 3D bioreactor for use in tissue engineering and regenerative medicine and the growth and proliferation of all types of cells in a pseudo-biological model of the bioreactor to provide tissues of several millimeters to several centimeters and use them in repairing all kinds of tissue defects.

Also, the practical goals of this project are as follows: commercialization of the research results in order to produce the mentioned biological reactor in pilot and industrial form, helping to solve the problem of tissue source for the repair of various types of tissue defects with bioreactor-scaffold cell culture, generalization of the results to Pre-clinical and clinical and providing the basis for the use of biological reactor in human tissue repair and generalizing the research results and using the bioreactor structure to obtain cells containing antibodies against cancer cells in the treatment of malignancies.

Solution of problem

In the last decade, many advances have been made in the evolution of cell culture technology. Stirred tank bioreactors evolved by providing a low-mobility environment with proper aeration and stirring systems. Cultivation systems for immobile cells such as hollow-fiber, fluidized-bed, and fixed-bed bioreactors were designed to protect cells from stress situations. With the invention of single-use reactors, new cultivation methods have become possible.

The main issue in the design and construction of a bioreactor with a fixed substrate is to create the necessary and sometimes competitive conditions for the cell culture process. These conditions include providing large amounts of oxygen needed by growing cells and in their high density, providing appropriate nutrients and protecting cell damage caused by high shear rates. Basically, all these bioreactors have a column of concentrated carrier material to support the growth of cells in a stationary state and a conditioning vessel containing modified and renewed culture medium. The medium moves in a rotating path from the fixed bed into the tank and is enriched with oxygen, while the attached and floating cells remain in the bed. Oxygenation is basically done by bubble aeration so that the cells are not in direct contact with the bubbles and therefore cell damage caused by air bubbles is not considered. In the tank, the consumed medium and the produced metabolites are exchanged. Normally, control elements such as O2, PH and temperature sensors are installed in the tank section to control the process.

As mentioned, the centrality of this bioreactor includes the use of a dense fixed bed with customized microcarriers, which will use exclusive hydrogel scaffolds in this project. This matrix contains a variety of cell adhesion molecules and creates a dense and large growth surface with cell penetration pores. Compared to other bioreactors, in this reactor, detailed and time-consuming processes such as manual procedures and step-by-step transfer from pre-cultivation to the final product are not performed. Because the multiplication and increase of biological mass is done in a fixed bed, this bioreactor can also work with low concentration cell injection, which simplifies the cultivation process and manual operations and subsequently reduces costs. The circulation of the liquid medium and its distribution in the same and uniform manner is created by a magnetic motor propeller that provides the lowest shear stress and maximum cell survival. The medium moves across the fixed bed from the bottom to the end and up, at the top this liquid falls into the medium tank as a thin layer, and in this way it is resaturated by oxygen to reach the highest limit of KLa O2 (mass transfer capacity oxygen). This unique way of oxygenation along with the complete mixing and immobility of the biological mass (scaffold) makes this bioreactor system create and maintain a high density of cells per unit volume. The simplicity of the process that continues from the injection of cells into the biomass to the production of the final product forms the central core of this platform. Therefore, the cells are immobile and fixed inside the fixed Bed and the system works as a perfusion model without the need to centrifuge the cultured cells.

Bioreactor components include: outer shell made of USP class VI hard plastic or glass, upper lid with different props and ports including media in/out - DO, PH & T props - Gas in/out sampling port and NaoH port, container containing fixed bed filled with scaffold or microcarrier, chamber containing mixing system including magnetic impeller, gas needs including oxygen, carbon dioxide, air with one pressure, equipment and measuring tools including PH probe, DO probe, Temp. probe that can be accessed as classic probes and peristaltic perfusion pump and NaoH if available (all equipment will be connected to their control systems.)

Preparing the scaffolding carrier:

The scaffold carrier will be obtained as a patent product from the sheep intestine in the form of a gel scaffold. In this way, we will obtain the intestinal submucosal scaffold (SIS) in the following 6 steps, which include:

Mechanical separation: We will obtain sheep intestine from animals up to 6 months old, weighing 80-100 kg and with the permission of the veterinary organization from the slaughterhouse. Immediately, the intestine (jejunum section) will be placed in phosphate buffer solution (PBS) containing 2% sterile penicillin-streptomycin solution and will be transferred to the laboratory, and after complete washing with PBS, it will be cut into 10 cm pieces and each piece from the middle as we cut it lengthwise and spread it. Then, the outer part, which contains the serous and muscular layer of the intestine, is completely separated with a blunt knife, and we do the same with the inner layer of the intestine (mucous layer) to finally obtain a membrane curtain, which is the layer under the intestinal mucosa.

Degreasing: In this step, the submucous membrane will be immersed in a methanol-chloroform solution with an equal volume of each for 12 hours under a chemical hood and then washed 3 times with deionized water.

Enzymatic digestion: the membrane will be incubated in 0.05% trypsin for 12 hours and then washed 3 times with 0.9% normal saline.

Cleaning and washing with: Sodium dodecyl sulfate (SDS) In this step, shake the obtained scaffold in 0.05% SDS solution for 6 hours using the incubator shaker until all the remaining cells of the intestinal tissue are removed and an Acellular scaffold is obtained.

Using the Freeze dryer for freezing and drying

And then the following steps are performed:

Grinding frozen SIS plates by freezer mill
Complete dissolution of SIS powder in an acid protease to produce digestion solution (3% acetic acid and 0.1% pepsin for 48 hours)
Raising the pH of the digestion solution to 7.8-7.2 by adding one mole of NaOH to create a neutral digestion solution
Making this solution in the form of a gel at a temperature of approximately 25°C (preferably). However, gelation progresses much faster at temperatures above 30°C and increases as it approaches the body's physiological temperature.

6- Sterilization: After obtaining the scaffold, it will be placed inside the fixed bed as a carrier and sterilized by ethylene oxide gas or UV rays.

After assembling and installing the cover, the inside of the bioreactor system compartment is washed with saline and washed again with PBS to remove the residual saline in the system wall, then we cover it with a layer of silicone oil to Prevent cells from sticking to places other than the fixed substrate. After this step, we will install all the probes and ports in their place and sterilize the device overnight using UV rays.

Next, fill the medium tank with 300-400 cc of DMEM culture medium and connect a 1500 cc DMEM reserve tank to the system through the Medium IN port.

All oxygen, carbon dioxide and air inlets will be connected to the corresponding port by connecting to the 0.2 micron microfilter. In order to create a homogeneous environment and the same temperature in the system, a 3 cm magnet was innovatively placed inside the mixing chamber, and the reactor system was operated on a shaker-heater device with an rpm of 0-1500 and an optimal temperature of 37 ± 0.5˚C.

PH, DO and temperature sensor props are connected to their control systems. By establishing a circulating flow inside the device, we allow the medium inside the fixed bed to move from the bottom to the top, so that finally a circuit of the medium flow from the fixed bed to the medium tank and vice versa is created. The initial density of the cells used in this system is 1 x 2-105 cells per ml and will continue for 1-2 weeks. During this period, sampling of the fixed substrate will be done at the end of the first and second week through the sampling port and will be evaluated in terms of histology and cell proliferation.

Isolation of stem cells:

The cells used in this bioreactor for growth and colonization will be the type of mesenchymal cells derived from adipose tissue, which are treated by a non-enzymatic method (an innovative method that is of therapeutic and clinical importance due to the absence of the use of enzymes in cell isolation) will be obtained and by performing flow cytometry, specific surface markers of these cells, i.e. CD109+, CD44+ and lack of expression of CD34+ marker, will be confirmed as markers of endothelial cells. After obtaining the desired volume of ASCs, they will be injected into the fixed bed cell through the cell port.

Advantage effects of invention

A container that controls the environment in relation to PH, temperature, dissolved oxygen and dissolved CO2 concentration.

Homogenize and mix the culture medium with minimal agitation and shear stress and aeration

Biomass is an effective heat conductor

Having a surface for cell attachment in the case of adherent cells

Ability to measure critical process and key parameters

Ability to increase scale

Durability and long-term sterilization

Easy to carry

Maintenance capability

: Bioreactor structure and performance overview

: Overview of the bioreactor

: Bottom view of the bioreactor

: Bioreactor structure and performance overview

: 1. Main or external shell 2. Inner shell 3. fixed porous substrate 4. Bottom pages 5. Rotor

Examples

This bioreactor includes the use of a dense fixed bed with customized micro carriers, which uses an exclusive hydrogel scaffold in this design. This matrix contains a variety of cell adhesion molecules and creates a dense and large growth surface with cell penetration pores. The simplicity of the process that continues from the injection of cells into the biomass to the production of the final product forms the central core and innovative part of this platform. Therefore, the cells are immobile and fixed inside the fixed bed and the system works as a perfusion model without the need to centrifuge the cultured cells.

This structure can be used in all laboratories and hospitals

Claims

The structure obtained from the bioreactor with fixed bed hydrogel is for wound healing. The main feature of this bioreactor is the constant exchange of the culture medium throughout the system, improving the mass transfer of nutrients and waste materials, as well as applying minimum shear stress to the cells.
According to claim 1, the cylindrical shape of the part where the scaffolding is placed is created by placing cylinders with different diameters. Also, for non-cylindrical scaffolds, by using a holder suitable for the shape of the scaffold and cylinder, the holder can be tested with any scaffold with different dimensions, geometry and materials.
According to claim 1, this structure causes a uniform flow to be created on the growth site of the cells, which prevents damaging stresses.
According to claim 2, the cell culture bioreactor is a dynamic and continuous system that has the ability to control the entry and exit of the culture medium liquid, install oxygen, carbon dioxide, pressure, temperature and dissolved oxygen sensors.
According to claim 4, in dynamic culture, the bioreactor performs suspension, cell growth and product formation well by mixing, homogenization, diffusion and dispersion, and food distribution in dynamic mode is more uniform and growth Cells are also better.
According to claim 1, this structure includes a perfusion bioreactor, which consists of a pump for continuous injection of the culture medium into the scaffolding network, a tank for maintaining the culture medium, and a holder that enables the installation of the scaffold.
According to claim 6, cell culture using a perfusion bioreactor causes a more uniform distribution of cells along the scaffold.
According to claim 6, the flow of the medium continues into the scaffold by means of a magnetic impeller. The position of the scaffold is kept in the central part of the system in the medium flow path.
According to claim 8, the culture medium flows into the injectable hydrogel scaffold obtained from the extracellular matrix of the sheep intestine, and as a result, fluid transfer increases.
According to claim 9, the use of the extracellular matrix of the sheep intestine in the form of a gel scaffold causes cell growth and colonization.
According to claim 1, the gel scaffold structure can be made in the form of all kinds of bone wounds, skin, vocal cords, esophageal tissue, bile ducts, tunica albuginea, kidney, ureter and abdominal wall and by stimulating the presence of cells from The surrounding tissues at the wound site accelerated the healing process of the defect.
According to claim 11, considering that all types of cells, including stem cells, have the ability to grow and multiply on the gel scaffold that can be injected in the bioreactor model, it is possible to obtain tissues of several millimeters to several centimeters needed for restoration. Acquired a variety of tissue defects.
According to claim 1, mesenchymal cells of different origins such as ADSCs can be used in this structure.
According to claim 13, mesenchymal stem cells derived from adipose tissue are isolated by a non-enzymatic method, these cells have the ability to be used in clinical trials.
In this structure, the stirrer speed can be adjusted, and the speed of the stirrer can be increased and decreased, and the separation of the scaffold holding chamber and the cells from the condition media ensures the safety of stress-sensitive biological cells.
The bioreactor has the ability to install a UV lamp, temperature gauge, flow meter, pressure gauge, speedometer and camera.
According to claim 1, the outer shell material can be changed from USP class VI hard plastic or glass to stainless steel. With the use of hard plastic equipment, it is possible to use it once in small volumes for clinical applications, and with the use of stainless steel equipment, it is possible to sterilize by thermal bioreactor method, and it is possible to use any type of three-dimensional scaffold. There are different dimensions and shapes to conduct studies in tissue engineering and stem cells in this bioreactor.