The creation of a cell-engineered pancreatic construct (CEPC) from islets of Langerhans and biocompatible matrix carrier (framework/scaffold), which imitates the native microenvironment of pancreatic tissue, is an approach to the treatment of type I diabetes mellitus (T1D).The objective of this work is to conduct a comparative analysis of the functional efficacy of CEPC and isolated rat islets of Langerhans after intraperitoneal administration into rats with experimental T1D.Materials and method. T1D was induced in rats by injecting low-dose (15 mg/ kg) streptozotocin (STZ) for 5 days. CEPC samples were created using viable and functional allogeneic isolated islets of Langerhans and tissue-specific scaffold obtained by decellularization of human pancreatic fragments. The rats received intraperitoneal injection of allogeneic islets of Langerhans (experimental group 1, n = 4) and CEPC (experimental group 2, n = 4). Control group rats received no treatment (n = 4). Blood glucose levels in the rats were measured, and the pancreas and kidneys of the experimental animals were examined histologically. The follow-up period for all animals continued for 10 weeks. Results. In experimental group 1, on day 7 after injection of Langerhans islets, glycemia decreased significantly from 28.2 ± 4.2 mmol/L to 13.4 ± 2.6 mmol/L. This fall persisted for 7 weeks, following which blood sugar increased to nearly their initial levels (prior to islets administration). In experimental group 2, on day 7 after CEPC administration, there was a more noticeable drop in blood sugar levels from 25.8 ± 5.1 mmol/L to 6.3 ± 2.7 mmol/L compared to experimental group 1. By the 10th week of the experiment, the average glucose level was two times lower than it was at the beginning. Blood glucose levels dropped more sharply in the CEPC group than in the islet group (by 75.6% and 52.5%, respectively).Conclusion. In T1D rats, CEPC has a more potent antidiabetic effect than islets of Langerhans. Thus, it has been shown that a tissue-specific scaffold may be used to create bioartificial pancreas in order to increase the functional efficiency of islets.

Objective: to obtain a stable mouse model of type 1 diabetes mellitus (T1DM) using streptozotocin (STZ), which has a toxic effect on pancreatic beta cells.Materials and methods. Experiments were performed on 30 white non-diabetic male mice of the SHK colony, which were injected intraperitoneally with STZ at a dose of 200 mg/ kg by two methods: 15 animals (group 1) once and 15 animals (group 2) intermittently – 5 consecutive days at 40 mg/kg per day.Results. In group 1, one mouse died after 2 days due to hypoglycemic coma, 4 mice developed hyperosmolar hyperglycemia (>33.3 mmol/l), 3 mice had spontaneous remission of diabetes, and 7 mice had stabilized hyperglycemia at levels close to 20 mmol/l. In group 2, only one mouse showed spontaneous remission of diabetes, while the remaining 14 animals showed stable diabetes with average hyperglycemia levels moderately above 20 mmol/L until the end of the 4-week follow-up. A histological study of the pancreas of these animals confirmed the destructive effect of STZ on islets in the form of mass death of insulin-producing β-cells.Conclusion. Split-dose intraperitoneal injection of STZ provides a stable experimental T1DM in 93% of laboratory mice.

Creation of a bioartificial pancreas, including a cell-engineered construct (CEC) formed from pancreatic islets (islets of Langerhans) and a biocompatible matrix mimicking the native microenvironment of pancreatic tissue, is one of the approaches to the treatment of type 1 diabetes mellitus (T1D). Objective: to conduct preliminary in vivo studies of the functional efficacy of intraperitoneal injection of a cell-engineered pancreatic endocrine construct and a suspension of rat pancreatic islets in an experimental T1D model. Materials and methods. Tissue-specific scaffold was obtained by decellularization of human pancreatic fragments. The viability and functional activity of rat islets isolated with collagenase were determined. Experimental T1D was modeled by intraperitoneal injection of low-dose streptozotocin and incomplete Freund's adjuvant into rats. The rats were intraperitoneally injected twice with pancreatic CEC (n = 2) or islet suspension (n = 1). Glucose levels in the blood and urine of the rats were assessed. Histological examination of organs (pancreas and kidneys) of the experimental animals was carried out. Results. After the first injection, blood glucose levels gradually decreased in all animals by more than 47% of the initial values; by follow-up day 24, the glucose level rose to the initial hyperglycemic values. After repeated administration, a 63.4% decrease in glycemic level was observed in the rats with pancreatic CEC and a 47.5% decrease in the one with islet suspension. At week 5 of the experiment, blood glucose levels gradually increased in all animals. At the same time, the glycemic index of the rat with injected pancreatic CEC was 62% lower than the glycemic index of the rat with injected islets. Conclusion. Allogeneic pancreatic islets in pancreatic CEC increase the duration of stable glycemic level in T1D rats.

The success of pancreatic islet allotransplantation in the treatment of patients with a difficult-to-manage type 1 diabetes depends mainly on the quantity and quality of islets isolated from the pancreas of deceased donors using enzyme preparations, primarily collagenase. Numerous studies on improvement and standardization of islet isolation techniques have reached their limits in the last decade. This has made it impossible to further boost the number and quality of clinical transplants. Taking into account the negative impact of collagenase technique on the morphofunctional properties of isolated islets, this work has studied the possibility of enzyme-free isolation of islet tissue purified of exocrine ballast. Experiments using the pancreas of newborn and young rabbits showed that developing methodological approaches to obtaining islet-like cultures without the use of exogenous enzymes is feasible.

Introduction. Developing a tissue-engineered pancreatic construct (TEPC) involves a search for matrices/scaffolds capable of mimicking the structure and composition of the natural extracellular matrix (ECM), which is an important component of the tissue microenvironment. A cell-free, tissue-specific matrix obtained from pancreas decellularization seems to be the most suitable for creation of a TEPC. The choice of pancreatic tissue decellularization protocol should take into account the morphological characteristics of the original pancreas. Preservation of the architectonics and composition of the native tissue in the decellularized pancreas matrix (DPM), and the presence of native ECM components allow for creation of conditions for prolonged vital activity of functionally active islet (insulin-producing) cells when creating TEPC. Objective : to determine the optimal parameters for decellularization of deceased donor pancreas with fibrosis, lipomatosis, and without pronounced signs of fibrosis and lipomatosis. Materials and methods . We used the caudal part of the pancreas obtained after multiorgan procurement from deceased donors, which was unsuitable for transplantation. Tissue-specific matrix was obtained by a combination of physical and chemical methods of pancreatic decellularization. A freeze-thaw cycle protocol and two protocols using osmotic shock were used. Samples of initial pancreatic tissue and decellularized fragments were subjected to histological analysis. Result s. It was shown that a physico-chemical method with freeze-thaw cycles is suitable for effective pancreatic decellularization in severe lipomatosis; a physico-chemical method using osmotic shock, but different protocol variants, is suitable for pancreas with diffuse fibrosis and for pancreas without pronounced signs of fibrosis and lipomatosis. Conclusion . For complete human pancreatic decellularization, the protocol should be correlated with histological features of the original tissue.

One of the pressing issues in tissue engineering is on how to obtain an artificial matrix that can simulate a biological microenvironment for cells. When creating a bioengineered pancreatic construct, a tissue-specific scaffold obtained from decellularized pancreatic tissue can serve as such matrix. Objective: to obtain and study the characteristic properties of a tissue-specific pancreas scaffold from decellularized human pancreatic fragments. Materials and methods. The decellularization protocol included 3 freeze/thaw cycles, followed by treatment with surfactants (sodium dodecyl sulfate and Triton X100). At each decellularization stage, samples were routinely stained with hematoxylin and eosin and for total collagen. In addition, immunohistochemical staining of decellularized human pancreas (DHP) for type I collagen and elastic fibers was performed. Cell nuclei in the original samples and the resulting matrix were visualized using DAPI fluorescent staining. DNA quantity in the native and decellularized pancreatic tissue was determined. The cytotoxicity of the tissue-specific matrix was evaluated in vitro by direct contact. The matrix properties of DHP samples were determined using mesenchymal stem cells (MSCs) of human adipose tissue. Results. A pancreatic decellularization method is proposed. This method allows to obtain a tissue-specific matrix in the form of a connective tissue scaffold completely free of detritus with preserved thin-fiber mesh-like structure, in which elastic and collagen fibers, including type I collagen, are identified. DAPI staining confirmed the absence of nuclear material in the decellularized matrix, while residual amount of DNA did not exceed 0.1%. Absence of matrix cytotoxicity and its ability to maintain adhesion and proliferation of human adipose tissue-derived MSCs was proved. Conclusion. As one of the stages in creating a bioengineered pancreatic construct, a method has been developed for producing a biocompatible (lack of cytotoxicity and immunogenicity) tissue-specific scaffold from decellularized human pancreatic tissue. In the scaffold, the morphofunctional properties of the native extracellular matrix-based scaffolds of the pancreas are preserved. Adhesion and proliferation of cell cultures are ensured.

One of the pressing issues in tissue engineering is on how to obtain an artificial matrix that can simulate a biological microenvironment for cells. When creating a bioengineered pancreatic construct, a tissue-specific scaffold obtained from decellularized pancreatic tissue can serve as such matrix. Objective : to obtain and study the characteristic properties of a tissue-specific pancreas scaffold from decellularized human pancreatic fragments. Materials and methods . The decellularization protocol included 3 freeze/thaw cycles, followed by treatment with surfactants (sodium dodecyl sulfate and Triton X100). At each decellularization stage, samples were routinely stained with hematoxylin and eosin and for total collagen. In addition, immunohistochemical staining of decellularized human pancreas (DHP) for type I collagen and elastic fibers was performed. Cell nuclei in the original samples and the resulting matrix were visualized using DAPI fluorescent staining. DNA quantity in the native and decellularized pancreatic tissue was determined. The cytotoxicity of the tissue-specific matrix was evaluated in vitro by direct contact. The matrix properties of DHP samples were determined using mesenchymal stem cells (MSCs) of human adipose tissue. Results . A pancreatic decellularization method is proposed. This method allows to obtain a tissue-specific matrix in the form of a connective tissue scaffold completely free of detritus with preserved thin-fiber mesh-like structure, in which elastic and collagen fibers, including type I collagen, are identified. DAPI staining confirmed the absence of nuclear material in the decellularized matrix, while residual amount of DNA did not exceed 0.1%. Absence of matrix cytotoxicity and its ability to maintain adhesion and proliferation of human adipose tissue-derived MSCs was proved. Conclusion . As one of the stages in creating a bioengineered pancreatic construct, a method has been developed for producing a biocompatible (lack of cytotoxicity and immunogenicity) tissue-specific scaffold from decellularized human pancreatic tissue. In the scaffold, the morphofunctional properties of the native extracellular matrix-based scaffolds of the pancreas are preserved. Adhesion and proliferation of cell cultures are ensured.

Objective : to study the effect of intrasplenic implantation of a tissue-engineered pancreatic construct (TEPC) on experimental diabetes mellitus. Materials and methods . Floating islet-like cultures (FICs) were obtained from the pancreas of newborn rabbits. To form TEPC, FICs were incubated with biopolymer microheterogeneous collagen-containing hydrogel (BMCH). TEPC samples were injected into the splenic pulp of rats with streptozotocin-induced diabetes. Results . TEPC with insulin-producing activity was formed on the 7–10th day of incubation of FICs with BMCH. After TEPC implantation in recipient rats, persistent decrease in hyperglycemia and disappearance of clinical signs of diabetes were noted. Histological analysis revealed the presence of groups of islet cells without signs of immune cell response at the TEPC implantation site. Conclusion . Our findings indicate that xenogeneic islet cells that were part of the TEPC of the pancreas can survive and actively function after implantation in the splenic pulp of diabetic rat.

Aim: to study the effect of implantation of tissue-engineering pancreatic construct (TEPC) on the course of experimental diabetes mellitus.Materials and methods. The TEPC samples received as a result of joint incubation in vitro floating islet-like cultures (FILC), obtained from the pancreas of newborn rabbits, and the biopolymer microheterogeneous collagen hydrogel (BMCH). Stable diabetes mellitus was caused in Wistar rats by the method of fractional streptozotocin administration.Results. The formation of TEPC occurred at 7-10 days of incubation FILC with BMCH. At the same time, the presence of β-cells with insulin-producing activity was revealed in the TEPC. After intraperitoneal implantation of TEPC samples in rats with streptozotocin diabetes mellitus, there was a significant and persistent decrease in glycemia until the end of the 8-week period of the experiment. Morphological study of pancreas of recipient rats revealed signs of regeneration of own β-cells.Conclusion. The data obtained suggest the combined antidiabetic effect of intraperitoneal injection of TEPC, due to both the direct functioning of the implant and its stimulating effect on the regeneration of β-cells in their own islets of rats with streptozotocin diabetes mellitus.

Introduction. Creation of a biomedical cell product – a bioengineered pancreatic construct – is hampered by problems associated with maintaining the viability of functionally active isolated islets of Langerhans (ILs). Both biopolymer and tissue-specific scaffolds can contribute to maintaining the structure and function of isolated ILs in vitro and in vivo. The most preferred tissue-specific scaffolds for cells can be obtained via decellularized pancreas matrix scaffold (DP matrix scaffold). Objective: to conduct a comparative analysis of the secretory function of isolated ILs of rats cultured in biopolymer-based collagen-containing hydrogel (BCH) and tissue-specific DP matrix scaffold, respectively. Materials and methods. ILs from rat pancreas was isolated using classical collagenase technique with some modifications. ILs were cultured in BCH and tissue-specific scaffold under standard conditions. Tissue-specific DP matrix scaffold was obtained through decellularization of rat pancreas. The DP matrix scaffold was examined for cytotoxicity and DNA presence; it was subjected to morphological study. The secretory function of ILs was studied through enzyme-linked immunosorbent assay (ELISA). Results. The secretory function of islets cultured in BCH and DP scaffolds is significantly higher than in the monoculture of islets. The advantage of using tissue-specific DP matrix scaffolds when creating bioengineered constructs of the pancreas over BCH matrix scaffolds was identified. Conclusion. BCH and tissue-specific DP scaffolds contribute not only to preserving the viability of isolated ILs, but also to prolonging their secretory capacity for 10 days, compared with ILs monoculture.

Introduction. Modern techniques of tissue engineering in the treatment of some degenerative diseases suggest the prospective viability of the biomedical technologies based on the creation of the equivalent of the damaged tissue (organ), including the tissue-engineered construct (TEC) of the endocrine pancreas (EP). Obtaining viable islets of Langerhans (IL) from the pancreas is a decisive step towards the creation of a TEC EP. The classic method of IL separation is based on enzymatic digestion of pancreatic tissue and further islet purification in ficoll density gradient during centrifugation, which adversely affects the morphofunctional state of IL.The aim of the study was the development of a method for separating viable pancreatic islets from a fragment of human pancreatic tail with different cold ischemia times.Materials and methods. A procedure of IL separation is proposed to be conducted without the use of EP tissue collagenase perfusion in the Ricordi chamber at the stage of IL separation and without ficoll solution with a varying density gradient at the stage of IL purification. Identification of IL obtained was performed by dithizone staining. The IL viability was evaluated using the LIVE/DEAD ® Cell Viability Kit. Histological analysis of the initial material included routine staining methods as well as immunohistochemical staining of the main types of islet cells.Results. The morphological study of the EP fragments at different times of cold ischemia did not reveal significant differences in the histological presentation of the organ parenchyma; the islet structure appeared intact. Vital staining confirmed the separated IL viability in vitro for at least 1–3 days.Conclusion. The proposed method of pancreatic tissue treatment allowed to reduce the number of stages, thereby minimizing the adverse effects of centrifugation and ficoll on the integrity of IL. It is possible to obtain the necessary amount of viable IL from a small EP fragment with the cold ischemia time of up to 19 hours, which can be used to create a TEC of a pancreas.

Aim:to develop methods for obtaining islet cell cultures for the purpose of their further use as a suitable component of the pancreatic tissue engineered construct. As a source of islet cell cultures, pancreas of newborn rabbits was used as an accessible and well-studied donor model.Materials and methods.For the obtaining of islet cell cultures, pancreas of 1–3-day-old newborn rabbits were used. Changes occurring during the cultivation of pancreatic tissue were recorded using an inverted microscope and a biostation. Morphological analysis of culture samples was carried out using histological and specifi c immunohistochemical methods. The insulin-producing activity of the cultures was determined by enzyme immunoassay.Results.Three main types of cultures were obtained: isletlike organotypic, suspensionalcytotypic, and monolayered, consisting of progenitor cells. Greater morphological safety and adequate insulin-producing ability was revealed in fl oating islet-like cultures.Conclusion.According to their morphofunctional properties, fl otation islet-like cultures obtained from pancreas of the newborn rabbits can be used as basal cell component of the experimental model of the tissue engineered construct of the pancreas.

Introduction. Extracellular matrix play an essential role in providing structural integrity and physiological support to Langerhans islets in pancreas. Imitation of the native microenvironment can be useful for viability of isolated pancreatic islets in vitro and in vivo. Aim. The purpose of this study was to characterize and compare the effect of biopolymer microstructured collagen-containing hydrogel (BMCH) on isolated rat islets survival. Materials and methods. Islets were isolated by classic collagenase techniques with some modifications. There were used hystological, immunofluorescence and immunohystochemistry methods. Results. Rat islets cultured with collagen-based gel don’t revealed destructive changes of structure and remained viabile 7 days incubation. Conclusion. Positive effect of BMCH to rat islet survival was revealed.

The aim of the work was detailed morphological investigations of donor pancreas (DP) for the study of possibilities of maximal selection of islet tissue suitable for transplantation to a patient of diabetes mellitus type 1. Materials and methods. Eight DPs were received as a result of multiorgan donation. Morphological investigations were performed by means of histological and special immunohistochemical methods. Results. The Majority of islets were revealed in the tail part of the DP. Besides typical Langerhans islets with predominance of mosaically located beta cells, the accumulations of islet cells forming so-called interlobular (perilobular) islets were revealed in the layers of interlobular connecting tissue. In addition, in the cells of ductal epithelium nestin which is a marker of progenitor cells was revealed. Conclusion. To obtain the maximal potential of islet tissue from DP it is necessary to use interlobular located islets as well as to use progenitor cells of pancreas, which have the ability to transdifferentiate into islet cells.

Aim. A study of the influence of microstructured collagen hydrogel (biomatrix) on survival and growth charac- teristics of islet cell cultures at their co-incubation. Materials and methods. As a biomatrix, the microstructured collagen hydrogel of linear series Sphero® GEL was used. Islet cell cultures obtained from newborn rabbit pan- creas were inoculated onto the biomatrix surface, covered with growth medium, and placed in a CO2 incubator. Changes occurring with biomatrix and cultures were observed by means of an inverted microscope and histolo- gical studies, including immunohistochemical analysis. Results. The presence of the microstructured collagen hydrogel matrix during the incubation of floating islet cell cultures promoted long-term preservation of the structural integrity and hormonal activity. Simultaneously the formation of cultures of pancreatic progenitor cells (islet cells precursors) was observed. Conclusion. Collagen hydrogel has a favorable effect on the formation and survival of islet cell cultures and can be used as a matrix of a tissue-engineered pancreas construct.

Кирсанова Л.А.1, Баранова Н.В.1, Бубенцова Г.Н.1, Скалецкая Г.Н.1, Перова Н.В.2, Севастьянов В.И.2 , Скалецкий Н.Н.1 1 Лаборатория клеточной трансплантации ФГБУ «Федеральный научный центр трансплантологии и искусственных органов им. ак. В.И. Шумакова» Минздрава РФ, Москва, Российская Федерация 2 Лаборатория тканевой инженерии и систем доставки отдела биомедицинских технологий и тканевой инженерии ФГБУ «Федеральный научный центр трансплантологии и искусственных органов им. ак. В.И. Шумакова» Минздрава РФ, Москва, Российская Федерация

Purpose. A comparative morphological analysis of adult pancreas and newborn rabbits as acceptable model for obtaining of islet cell cultures having a low immunogenicity was agoal of this study. Materials and methods. Pancreas from adult and newborn rabbits and islet cell culture was examined by histological and immunohistochemical techniques. Results. Shown, the pancreas of adult rabbits contains great amount of exocrine tissue and culturing it does not allow to obtain the purified islets of impurities. By contrast, pancreas of newborn rabbits in which the ratio of the islets and the exocrine tissue is much higher, it is possible to obtain highly purified cultures of islet cells. Conclusion. Morphological features of newborn rabbit pancreas can use it as a model for obtaining cultures of islet cells having low immunogenicity.