Uniform Cell Distribution Research Articles

Micro-Nanostructures of Cellulose-Collagen for Critical Sized Bone Defect Healing.

Bone tissue engineering strategies utilize biodegradable polymeric matrices alone or in combination with cells and factors to provide mechanical support to bone, while promoting cell proliferation, differentiation, and tissue ingrowth. The performance of mechanically competent, micro-nanostructured polymeric matrices, in combination with bone marrow stromal cells (BMSCs), is evaluated in a critical sized bone defect. Cellulose acetate (CA) is used to fabricate a porous microstructured matrix. Type I collagen is then allowed to self-assemble on these microstructures to create a natural polymer-based, micro-nanostructured matrix (CAc). Poly (lactic-co-glycolic acid) matrices with identical microstructures serve as controls. Significantly higher number of implanted host cells are distributed in the natural polymer based micro-nanostructures with greater bone density and more uniform cell distribution. Additionally, a twofold increase in collagen content is observed with natural polymer based scaffolds. This study establishes the benefits of natural polymer derived micro-nanostructures in combination with donor derived BMSCs to repair and regenerate critical sized bone defects. Natural polymer based materials with mechanically competent micro-nanostructures may serve as an alternative material platform for bone regeneration.

Modified conventional bioreactor for microalgae cultivation

Microalgae, a renewable source for third generation biofuel production, have a great potential if cultivated in high concentration economically. Bottleneck lies with designing economical and efficient photobioreactor. In addition, proportional C and N inputs in the known media does not support high specific growth rate and high biomass build-up. Nitrates in fermentation media, f/2 for Nannochloropsis sp. and Zarrouk's for Arthrospira platensis, were modified. Aeration and agitation were altered in conventional bioreactor (BIOFLO 110) to reduce power consumption, increase mixing time and prevents settling. This was achieved by introducing four way flow regime supporting uniform nutrient and cell distribution in media. Volumetric cell productivity for Nannochloropsis sp. and A.platensis were achieved as 0.618g/l/d and 0.774g/l/d, respectively. This photobioreactor also supported the maximum specific CO2 sequestration rates to the level of 0.42g/g/h and 0.39g/g/h for Nannochloropsis sp. and A.platensis, respectively, confirming efficient and effective operation.

Biocomposite nanofiber matrices to support ECM remodeling by human dermal progenitors and enhanced wound closure

Cell-based therapies have recently been the focus of much research to enhance skin wound healing. An important challenge will be to develop vehicles for cell delivery that promote survival and uniform distribution of cells across the wound bed. These systems should be stiff enough to facilitate handling, whilst soft enough to limit damage to newly synthesized wound tissue and minimize patient discomfort. Herein, we developed several novel modifiable nanofibre scaffolds comprised of Poly (ε-caprolactone) (PCL) and gelatin (GE). We asked whether they could be used as a functional receptacle for adult human Skin-derived Precursor Cells (hSKPs) and how naked scaffolds impact endogenous skin wound healing. PCL and GE were electrospun in a single facile solvent to create composite scaffolds and displayed unique morphological and mechanical properties. After seeding with adult hSKPs, deposition of extracellular matrix proteins and sulphated glycosaminoglycans was found to be enhanced in composite grafts. Moreover, composite scaffolds exhibited significantly higher cell proliferation, greater cell spreading and integration within the nanofiber mats. Transplantation of acellular scaffolds into wounds revealed scaffolds exhibited improvement in dermal-epidermal thickness, axonal density and collagen deposition. These results demonstrate that PCL-based nanofiber scaffolds show promise as a cell delivery system for wound healing.

Semi-automated scoring of triple-probe FISH in human sperm using confocal microscopy.

Structural and numerical sperm chromosomal aberrations result from abnormal meiosis and are directly linked to infertility. Any live births that arise from aneuploid conceptuses can result in syndromes such as Kleinfelter, Turners, XYY and Edwards. Multi-probe fluorescence in situ hybridization (FISH) is commonly used to study sperm aneuploidy, however manual FISH scoring in sperm samples is labor-intensive and introduces errors. Automated scoring methods are continuously evolving. One challenging aspect for optimizing automated sperm FISH scoring has been the overlap in excitation and emission of the fluorescent probes used to enumerate the chromosomes of interest. Our objective was to demonstrate the feasibility of combining confocal microscopy and spectral imaging with high-throughput methods for accurately measuring sperm aneuploidy. Our approach used confocal microscopy to analyze numerical chromosomal abnormalities in human sperm using enhanced slide preparation and rigorous semi-automated scoring methods. FISH for chromosomes X, Y, and 18 was conducted to determine sex chromosome disomy in sperm nuclei. Application of online spectral linear unmixing was used for effective separation of four fluorochromes while decreasing data acquisition time. Semi-automated image processing, segmentation, classification, and scoring were performed on 10 slides using custom image processing and analysis software and results were compared with manual methods. No significant differences in disomy frequencies were seen between the semi automated and manual methods. Samples treated with pepsin were observed to have reduced background autofluorescence and more uniform distribution of cells. These results demonstrate that semi-automated methods using spectral imaging on a confocal platform are a feasible approach for analyzing numerical chromosomal aberrations in sperm, and are comparable to manual methods. © 2017 International Society for Advancement of Cytometry.

Comparison of conventional smear cytology and manual liquid based cytology based on smears from normal oral exfoliated cells

Liquid-based cytology (LBC), since its inception in the 1990s, has shown significant advantages over conventional exfoliative cytology. LBC gives better results than conventional method as it not only enhances both sensitivity and specificity, but also provides material for further investigations. However, LBC requires expensive automated devices and materials, which might not be affordable for many cytopathology laboratories in countries with poor resources. In order to formulate an economic cytological procedure with the accuracy of LBC, smears made out of manual liquid based cytology (MLBC) technique was followed in this study and the results were compared with respective conventional cytology smears. To the authors’ knowledge, this is the first comparative study between MLBC and conventional method done for oral cavity in normal buccal mucosa. One hundred apparently normal subjects who reported to the outpatient department were selected after obtaining informed consent from all the subjects. Two Scrapings were made in each patient from the clinically normal appearing buccal mucosa. Both conventional and MLBC smears were stained by routine Papanicolaou technique and evaluated by three independent observers for specimen adequacy, thickness of the smear, uniformity of cell distribution, clarity or resolution of the cells, presence of background artifacts, and staining characteristics of the cells. Each parameter was graded as satisfactory; satisfactory but limited; or unsatisfactory. Number of satisfactory results obtained in MLBC smears was significantly higher than that by conventional smears. After comparison of smears prepared from conventional cytology and manual liquid-based cytology, we conclude that manual liquid-based cytology is both cost effective and also technically sound method with specific and clear results.

Effects of foaming parameters on microstructure and compressive properties of aluminum foams produced by powder metallurgy method

Semi open-cell aluminum foams having channels between individual cells were produced using low cost CaCO3 foaming agent and applying the powder compact melting process. To this end, the aluminum and CaCO3 powder mixtures were cold compacted into dense cylindrical precursors for foaming at specific temperatures under air atmosphere. The effects of several parameters including precursor compaction pressure, foaming agent content as well as temperature and time of the foaming process on the cell microstructure, linear expansion, relative density and compressive properties were investigated. A uniform distribution of cells with sizes less than 100 μm, which form semi open-cell structures with relative densities in the range of 55.4%–84.4%, was obtained. The elevation of compaction pressure between 127–318 MPa and blowing agent up to 15% (mass fraction) led to an increase in the linear expansion, compressive strength and densification strain. By varying the foaming temperature from 800 to 1000 °C, all of the investigated parameters increased except compressive strength and relative density. The results indicated the optimal foaming temperature and time as 900 °C and 10–25 min, respectively.

Facile fabrication of poly(ε-caprolactone)/graphene oxide membranes for bioreactors in tissue engineering

Promising polymer membranes of blended biocompatible poly(ε-caprolactone) and graphene oxide (PCL/GO) and PCL and partially reduced graphene oxide (PCL/rGO) with outstanding water and nutrient transport properties for cell culture bioreactors were prepared using phase inversion at mild temperatures. Some of the prepared PCL/GO membranes were subjected to a ‘chemical-free’ GO post-reductive process using UV (PCL/GO/UV) irradiation. The PCL/rGO membranes exhibited 2.5 times higher flux than previously reported biocompatible polymer membranes for cell culture bioreactors, which was attributed to the highly interconnected porosity. On the other hand, the formation of PCL-graphene oxide composites in the PCL/GO and PCL/GO/UV membranes was not conclusive according to spectroscopic analyses, thermal analyses and mechanical characterization, probably due to the low graphene oxide loading in the membranes (0.1%w/w). The presence of graphene oxide-based nanomaterials in the polymer matrix slightly reduced the mechanical properties of the PCL-graphene oxide membranes by limiting the polymer chain mobility in comparison to that of the plain PCL membranes. However, their mechanical stability was sufficient for the applications pursued. Finally, the biocompatibility assay indicated that the incorporation of GO and rGO into the PCL matrix enhanced the uniform distribution and morphology of the glioblastoma cells on the surface of the PCL-graphene oxide membranes.

Bioprinting multidimensional constructs: a quantitative approach to understanding printed cell density and redistribution phenomena

Currently, bioprinting high cellular fractions in bio-inks represents a critical step towards engineered tissue models with enhanced biological relevance. However, precise spatiotemporal mapping of high cell densities within hydrogel materials represents a significant challenge. To address these needs and challenges, this paper advances a systematic multidimensional analytical framework to quantitatively determine the spatial variations in cell density and temporal evolution of cell redistribution phenomena within bioprinted cell-laden fiber-based constructs as a function of the complex interplay of the various processing parameters for an alginate-gelatin hydrogel material system. For a one dimensional analysis, fibers printed at high relative viscosities exhibit large contact angles in the cross-sectional plane with a high degree of cell dispersion (Dd = 96.8 ± 6.27%). Conversely, fibers printed at low relative viscosities exhibit small contact angles with cells coalescing towards the fiber midline (Dd = 76.3 ± 4.58%). The observed in-process redistribution of cells is attributed to the flow topology inside the needle tip. The precise mechanism is governed by the temperature-dependent viscoplastic material properties as demonstrated numerically using the Herschel–Bulkley model. In the 2D analysis, the printed grid structures yield differences in local cell densities between the strut and cross regions within the fibers. At low relative viscosities, cells aggregate in regions where two overlapping fibers fuse together, yielding a high cell density ratio (1.79 ± 0.04) between the cross and strut regions. However, at high relative viscosities, the cell density ratio decreases (0.96 ± 0.03). In the 3D analysis, due to gravity and extrusion process effects, initially uniform bio-ink cell distributions within bio-inks are redistributed as a function of printing time, yielding quantitative increases in cell density for the initial layers, followed by quantitative decreases in the subsequent layers. Finally, a time course incubation study of the 3D bioprinted cell-laden construct reveals a time-dependent increase in cell density owing to proliferation that accelerates the material degradation.

Pattern formation in multiphase models of chemotactic cell aggregation.

We develop a continuum model for the aggregation of cells cultured in a nutrient-rich medium in a culture well. We consider a 2D geometry, representing a vertical slice through the culture well, and assume that the cell layer depth is small compared with the typical lengthscale of the culture well. We adopt a continuum mechanics approach, treating the cells and culture medium as a two-phase mixture. Specifically, the cells and culture medium are treated as fluids. Additionally, the cell phase can generate forces in response to environmental cues, which include the concentration of a chemoattractant that is produced by the cells within the culture medium. The model leads to a system of coupled nonlinear partial differential equations for the volume fraction and velocity of the cell phase, the culture medium pressure and the chemoattractant concentration, which must be solved subject to appropriate boundary and initial conditions. To gain insight into the system, we consider two model reductions, appropriate when the cell layer depth is thin compared to the typical length scale of the culture well: a (simple) 1D and a (more involved) thin-film extensional flow reduction. By investigating the resulting systems of equations analytically and numerically, we identify conditions under which small amplitude perturbations to a homogeneous steady state (corresponding to a spatially uniform cell distribution) can lead to a spatially varying steady state (pattern formation). Our analysis reveals that the simpler 1D reduction has the same qualitative features as the thin-film extensional flow reduction in the linear and weakly nonlinear regimes, motivating the use of the simpler 1D modelling approach when a qualitative understanding of the system is required. However, the thin-film extensional flow reduction may be more appropriate when detailed quantitative agreement between modelling predictions and experimental data is desired. Furthermore, full numerical simulations of the two model reductions in regions of parameter space when the system is not close to marginal stability reveal significant differences in the evolution of the volume fraction and velocity of the cell phase, and chemoattractant concentration.

Solutions of nonlinear differential equations with feature detection using fast Walsh transforms

Walsh functions form an orthonormal basis set consisting of square waves. Square waves make the system well suited for detecting and representing functions with discontinuities. Given a uniform distribution of 2p cells on a one-dimensional element, it is proved that the inner product of the Walsh Root function for group p with every polynomial of degree ≤(p−1) across the element is identically zero. It is also proved that the magnitude and location of a discontinuous jump, as represented by a Heaviside function, are explicitly identified by its Fast Walsh Transform (FWT) coefficients. These two proofs enable an algorithm that quickly provides a Weighted Least Squares fit to distributions across the element that include a discontinuity. It is shown that flux reconstruction relative to the FWT fit in partial differential equations provides improved accuracy. The detection of a discontinuity further enables analytic relations to locally describe its evolution and provide increased accuracy. Examples are provided for time-accurate advection, Burgers' equation, and quasi-one-dimensional nozzle flow.

Development of a novel alginate-polyvinyl alcohol-hydroxyapatite hydrogel for 3D bioprinting bone tissue engineered scaffolds.

Three-dimensional printed biomaterials used as personalized tissue substitutes have the ability to promote and enhance regeneration in areas of defected tissue. The challenge with 3D printing for bone tissue engineering remains the selection of a material with optimal rheological properties for printing in addition to biocompatibility and capacity for uniform cell incorporation. Hydrogel biomaterials may provide sufficient printability to allow cell encapsulation and bioprinting of scaffolds with uniform cell distribution. In this study, a novel alginate-polyvinyl alcohol (PVA)-hydroxyapatite (HA) hydrogel formulation with optimal rheological properties for 3D bioprinting of mouse calvaria 3T3-E1 (MC3T3) cells into scaffolds of high shape fidelity has been developed. A systematic investigation was conducted to determine the effect of varying concentrations of alginate, phosphate, calcium, and the PVA-HA suspension in the formulation on the resulting viscosity and thus printability of the hydrogel. HA, the main mineral component in natural bone, was incorporated into the hydrogel formulation to create a favorable bone-forming environment due to its excellent osteoconductivity. Degradation studies in α-MEM cell culture media showed that the 3D printed alginate-PVA-HA scaffolds remained in-tact for 14 days. MC3T3 cells were well distributed and encapsulated throughout the optimal hydrogel formulation and expressed high viability through the completion of the 3D printing process. Thus, the development of this novel, osteoconductive, biodegradable, alginate-PVA-HA formulation and its ability to 3D bioprint tissue engineered scaffolds make it a promising candidate for treating personalized bone defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1457-1468, 2017.

Increased lipid accumulation and adipogenic gene expression of adipocytes in 3D bioprinted nanocellulose scaffolds

Compared to standard 2D culture systems, new methods for 3D cell culture of adipocytes could provide more physiologically accurate data and a deeper understanding of metabolic diseases such as diabetes. By resuspending living cells in a bioink of nanocellulose and hyaluronic acid, we were able to print 3D scaffolds with uniform cell distribution. After one week in culture, cell viability was 95%, and after two weeks the cells displayed a more mature phenotype with larger lipid droplets than standard 2D cultured cells. Unlike cells in 2D culture, the 3D bioprinted cells did not detach upon lipid accumulation. After two weeks, the gene expression of the adipogenic marker genes PPARγ and FABP4 was increased 2.0- and 2.2-fold, respectively, for cells in 3D bioprinted constructs compared with 2D cultured cells. Our 3D bioprinted culture system produces better adipogenic differentiation of mesenchymal stem cells and a more mature cell phenotype than conventional 2D culture systems.

A novel method for blood-typing using nitrocellulose.

Blood wicking in its steady-state form, i.e. the uniform distribution of blood cells in plasma, is completely different from that in its coagulated form on a porous surface like paper. The hydrophilic property of the cellulose leads to a significant wicking of the blood cells on paper fibers after rinsing with isotonic solution. The difference in the wicking length of the blood cells in steady state and that in the coagulated form could be considered as a criterion to recognize the blood type in a paper-based kit. However, owing to the molecular structure of the nitrocellulose, a better process occurs while separating the coagulated blood from the steady-state form of cells. Therefore, it is possible to use the nitrocellulose for the blood-typing kit which leads to a simpler way to diagnose a blood type. Two series of experiments were performed on nitrocellulose membrane. First, antibody solutions and blood samples were sequentially absorbed on nitrocellulose strips, allowed to interact, rinsed with an isotonic solution and distilled water, and image processing performed on a digital picture of the remaining blood cells. The efficiency of the agglutinated blood cell fixation was quantified by red color intensity. Then, it was demonstrated that there is no considerable difference in fixation of agglutinated blood cells with rinsing using isotonic and nonisotonic solutions. This fact can be a considerable advantage over paper since it can eliminate the probable mistake from using unisotonic solution for rinsing. Second, owing to the nonwicking property of the blood cells on the hydrophobic nitrocellulose fibers, we employed another diagnostic criterion and investigated nitrocellulose blood-typing prototypes. The nitrocellulose blood-typing kit provides more simple, sensitive and trustworthy assay for rapid blood typing in situations with no access to laboratory facilities.

Responses of MSCs to 3D Scaffold Matrix Mechanical Properties under Oscillatory Perfusion Culture.

Both fluid shear stress and matrix stiffness are implicated in bone metabolism and functional adaptation, but the synergistic action of these mechanical cues on the biological behaviors of mesenchymal stem cells (MSCs) is still not well-known. In the present work, a homemade oscillatory flow device was applied to investigate the effects of matrix stiffness on MSCs survival, distribution, and osteogenic differentiation in three-dimensional (3D) conditions. Furthermore, the flow field and cell growth in this bioreactor were theoretically simulated. The results demonstrated that oscillatory shear stress significantly increased the viability and distribution uniformity of MSCs throughout the scaffold after culture for 3 weeks. Compared to static culture, oscillatory shear stress could promote the collagen secretion, mineral deposits, and osteogenic differentiation of MSCs. The findings obtained from this work indicate that the oscillatory perfusion not only provides a higher survival rate and a more uniform distribution of cells but also facilitates osteogenic differentiation of MSCs. Oscillating perfusion bioreactor culture of MSCs in 3D scaffold with optimal matrix stiffness could offer an easy-to-use but efficient bioreactor for bone tissue engineering.

Re-epithelialization of whole porcine kidneys with renal epithelial cells

Decellularized porcine kidneys were recellularized with renal epithelial cells by three methods: perfusion through the vasculature under high pressure, perfusion through the ureter under high pressure, or perfusion through the ureter under moderate vacuum. Histology, scanning electron microscopy, confocal microscopy, and magnetic resonance imaging were used to assess vasculature preservation and the distribution of cells throughout the kidneys. Cells were detected in the magnetic resonance imaging by labeling them with iron oxide. Perfusion of cells through the ureter under moderate vacuum (40 mmHg) produced the most uniform distribution of cells throughout the kidneys.

Design of a Novel Method for the Spatial Distribution of Cells within a Porous Scaffold for Tissue Engineering Applications

Tissue engineering is rapidly progressing to provide complex, three-dimensional (3D) representations of human tissues that can be used for tissue replacement and/or to study tissue systems. Tissue engineering includes the addition of cells within 3D scaffolds, along with bioactive components, sometimes within a bioreactor. A major challenge in developing many tissue-engineered models is the ability to evenly distribute cells throughout a porous scaffold, in order to achieve good cell viability and growth. In this study, we created a 3D collagen-chitosan scaffold with specific properties to aid in seeding cells within the entire volume and investigated a dynamic method to seed cells within such scaffold. Based on the requirements for cell seeding, the scaffolds were less than 500 µm thick, had pore sizes greater than 50 µm and had a porosity of 50% or greater. Fibroblasts were used as model cells for this seeding method. To seed fibroblasts within the scaffold, we varied two design parameters: concentration of the collagen seeding solution and the centrifugal force used for cell seeding. We ranked the seeding efficiency, cell proliferation and distribution in order to choose the ideal cell seeding method. Results showed that seeding with a higher concentration (2 mg/ml) of collagen seeding solution and a lower centrifugation speed (259 ×g) was the optimal seeding method, resulting in 84% increase in cell proliferation and a more uniform cell distribution throughout the scaffold. Results from this study can be applied for seeding a variety of cell populations within porous scaffolds for tissue engineering applications.

Gelatin-based extracellular matrix cryogels for cartilage tissue engineering

Abstract In this study, gelatin-based cryogels were fabricated by mixing methacrylated gelatin (GelMA) with methacrylated hyaluronic acid (MeHA) or methacrylated chondroitin sulfate (MeCS) for cartilage tissue engineering. In vitro revealed that MeCS incorporated gelatin-based cryogel (G-MeCS) showed significant cartilaginous tissue stimulation. Furthermore, the cell-laden gelatin-based ECM cryogels were implanted into mouse subcutaneous tissue for 6 weeks and displayed uniform distribution of cells with normal phenotype maintenance. Finally, when these cryogels were implanted into osteochondral defect of New Zealand white rabbit, full integration with host tissue and increased cellularity were observed with G-MeCS cryogel.

Biomineralization Guided by Paper Templates.

This work demonstrates the fabrication of partially mineralized scaffolds fabricated in 3D shapes using paper by folding, and by supporting deposition of calcium phosphate by osteoblasts cultured in these scaffolds. This process generates centimeter-scale free-standing structures composed of paper supporting regions of calcium phosphate deposited by osteoblasts. This work is the first demonstration that paper can be used as a scaffold to induce template-guided mineralization by osteoblasts. Because paper has a porous structure, it allows transport of O2 and nutrients across its entire thickness. Paper supports a uniform distribution of cells upon seeding in hydrogel matrices, and allows growth, remodelling, and proliferation of cells. Scaffolds made of paper make it possible to construct 3D tissue models easily by tuning material properties such as thickness, porosity, and density of chemical functional groups. Paper offers a new approach to study mechanisms of biomineralization, and perhaps ultimately new techniques to guide or accelerate the repair of bone.

Engineered electrospun poly(caprolactone)/polycaprolactone-g-hydroxyapatite nano-fibrous scaffold promotes human fibroblasts adhesion and proliferation

Polycaprolactone (PCL)/hydroxyapatite nano-composites are among the best candidates for tissue engineering. However, interactions between nHAp and PCL are difficult to control leading to inhomogeneous dispersion of the bio-ceramic particles. Grafting of polymer chains at high density/chain length while promotes the phase compatibility may result in reduced HAp exposed surface area and therefore, bioactivity is compromised. This issue is addressed here by grafting PCL chains onto HAp nano-particles through ring opening polymerization of ε-caprolactone (PCL-g-HAp). FTIR and TGA analysis showed that PCL (6.9wt%), was successfully grafted on the HAp. PCL/PCL-g-HAp nano-fibrous scaffold showed up to 10 and 33% enhancement in tensile strength and modulus, respectively, compared to those of PCL/HAp. The effects of HAp on the in vitro HAp formation were investigated for both the PCL/HAp and PCL/PCL-g-HAp scaffolds. Precipitation of HAp on the nano-composite scaffolds observed after 15days incubation in simulated body fluid (SBF), as confirmed by scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX).Human fibroblasts were seeded on PCL, PCL/HAp and PCL/PCL-g-HAp scaffolds. According to MTT assay, the highest cell proliferation was recorded for PCL/PCL-g-HAp nano-composite, at all time intervals (1–21days, P<0.001). Fluorescent microscopy (of DAPI stained samples) and electron microscopy images showed that all nano-fibrous scaffolds (PCL, PCL/HAp, and PCL/PCL-g-HAp), were non-toxic against cells, while more cell adhesion, and the most uniform cell distribution observed on the PCL/PCL-g-HAp.Overall, grafting of relatively short chains of PCL on the surface of HAp nano-particles stimulates fibroblasts adhesion and proliferation on the PCL/PCL-g-HAp nano-composite.

Embryonic Stem Cells-loaded Gelatin Microcryogels Slow Progression of Chronic Kidney Disease.

Background:Chronic kidney disease (CKD) has become a public health problem. New interventions to slow or prevent disease progression are urgently needed. In this setting, cell therapies associated with regenerative effects are attracting increasing interest. We evaluated the effect of embryonic stem cells (ESCs) on the progression of CKD.Methods:Adult male Sprague–Dawley rats were subjected to 5/6 nephrectomy. We used pedicled greater omentum flaps packing ESC-loaded gelatin microcryogels (GMs) on the 5/6 nephrectomized kidney. The viability of ESCs within the GMs was detected using in vitro two-photon fluorescence confocal imaging. Rats were sacrificed after 12 weeks. Renal injury was evaluated using serum creatinine, urea nitrogen, 24 h protein, renal pathology, and tubular injury score results. Structural damage was evaluated by periodic acid-Schiff and Masson trichrome staining.Results:In vitro, ESCs could be automatically loaded into the GMs. Uniform cell distribution, good cell attachment, and viability were achieved from day 1 to 7 in vitro. After 12 weeks, in the pedicled greater omentum flaps packing ESC-loaded GMs on 5/6 nephrectomized rats group, the plasma urea nitrogen levels were 26% lower than in the right nephrectomy group, glomerulosclerosis index was 62% lower and tubular injury index was 40% lower than in the 5/6 nephrectomized rats group without GMs.Conclusions:In a rat model of established CKD, we demonstrated that the pedicled greater omentum flaps packing ESC-loaded GMs on the 5/6 nephrectomized kidney have a long-lasting therapeutic rescue function, as shown by the decreased progression of CKD and reduced glomerular injury.