Fabrication and Characterization Techniques of In Vitro 3D Tissue Models.

The culturing of cells in the laboratory under controlled conditions has always been crucial for the advancement of scientific research. Cell-based assays have played an important role in providing simple, fast, accurate, and cost-effective methods in drug discovery, disease modeling, and tissue engineering while mitigating reliance on cost-intensive and ethically challenging animal studies. The techniques involved in culturing cells are critical as results are based on cellular response to drugs, cellular cues, external stimuli, and human physiology. In order to establish in vitro cultures, cells are either isolated from normal or diseased tissue and allowed to grow in two or three dimensions. Two-dimensional (2D) cell culture methods involve the proliferation of cells on flat rigid surfaces resulting in a monolayer culture, while in three-dimensional (3D) cell cultures, the additional dimension provides a more accurate representation of the tissue milieu. In this review, we discuss the various methods involved in the development of 3D cell culture systems emphasizing the differences between 2D and 3D systems and methods involved in the recapitulation of the organ-specific 3D microenvironment. In addition, we discuss the latest developments in 3D tissue model fabrication techniques, microfluidics-based organ-on-a-chip, and imaging as a characterization technique for 3D tissue models.

Endothelial progenitor/stem cells in engineered vessels for vascular transplantation.

BACKGROUND: Dysfunction of blood vessel leads to aneurysms, myocardial infarction and other thrombosis conditions. Current treatment strategies are transplantation of blood vessels from one part of the body to other dysfunction area, or allogenic, synthetic. Due to shortage of the donor, painful dissection, and lack of efficacy in synthetic, there is a need for alternative to native blood vessels for transplantation. METHODS: Human umbilical-cord tissue obtained from the hospital with the informed consent. Umbilical-cord blood vessels were isolated for decellularization and to establish endothelial cell culture. Cultured cells were characterized by immunophenotype, gene expression and in vitro angiogenesis assay. Decellularized blood vessels were recellularized with the endothelial progenitors and Wharton jelly, CL MSCs (1:1), which was characterized by MTT, biomechanical testing, DNA content, SEM and histologically. Bioengineered vessels were transplanted into the animal models to evaluate their effect. RESULTS: Cultured cells express CD31 and CD14 determining endothelial progenitor cells (EPCs). EPCs expresses various factors such as angiopoitin1, VWF, RANTES, VEGF, BDNF, FGF1, FGF2, HGF, IGF, GDNF, NGF, PLGF, NT3, but fail to express NT4, EGF, and CNTF. Pro and anti-inflammatory cytokine expressions were noticed. Functionally, these EPCs elicit in vitro tube formation. Negligible DNA content and intact ECM confirms the efficient decellularization of tissue. The increased MTT activity in recellularized tissue determines proliferating cells and biocompatibility of the scaffolds. Moreover, significant (P < 0.05) increase in maximum force and tensile of recellularized biomaterial as compared to the decellularized scaffolds. Integration of graft with host tissue, suggesting biocompatible therapeutic biomaterial with cells. CONCLUSION: EPCs with stem cells in engineered blood vessels could be therapeutically applicable in vascular surgery.

Enhanced Biocorrosion Resistance and Cellular Response of a Dual-Phase High Entropy Alloy through Reduced Elemental Heterogeneity.

The leaching out of toxic elements from metallic bioimplants has serious repercussions, including allergies, peripheral neuritis, cancer, and Alzheimer's disease, leading to revision or replacement surgeries. The development of advanced structural materials with excellent biocompatibility and superior corrosion resistance in the physiological environment holds great significance. High entropy alloys (HEAs) with a huge compositional design space and outstanding mechanical and functional properties can be promising for bioimplant applications. However, microstructural heterogeneity arising from elemental segregation in these multiprinciple alloy systems is the Achilles heel in the development of next-generation HEAs. Here, we demonstrate a pathway to homogenize the microstructure of a biocompatible dual-phase HEA, comprising refractory elements, namely, MoNbTaTiZr, through severe surface deformation using stationary friction processing (SFP). The strain and temperature field during processing homogenized the elemental distribution, which was otherwise unresponsive to conventional annealing treatments. Nearly 15 min of the SFP treatment resulted in a significant elemental homogenization across dendritic and interdendritic regions, similar to a week-long annealing treatment at 1275 K. The SFP processed alloy showed a nearly six times higher biocorrosion resistance compared to its as-cast counterpart. X-ray photoelectron spectroscopy was used to investigate the nature of the oxide layer formed on the specimens. Superior corrosion behavior of the processed alloy was attributed to the formation of a stable passive layer with zirconium oxide as the primary constituent and higher hydrophobicity. Biocompatibility studies performed using the human mesenchymal stem cell line, showed higher viability for the processed HEA compared to its as-cast counterpart as well as conventional metallic biomaterials including stainless steel (SS316L) and titanium alloy (Ti6Al4V).

Biocompatible High Entropy Alloys with Excellent Degradation Resistance in a Simulated Physiological Environment.

Bioimplants are susceptible to simultaneous wear and corrosion degradation in the aggressive physiological environment. High entropy alloys with equimolar proportion of constituent elements represent a unique alloy design strategy for developing bioimplants due to their attractive mechanical properties, superior wear, and corrosion resistance. In this study, the tribo-corrosion behavior of an equiatomic MoNbTaTiZr high entropy alloy consisting of all biocompatible elements was evaluated and compared with 304 stainless steel as a benchmark. The high entropy alloy showed a low wear rate and a friction coefficient as well as quick and stable passivation in simulated body fluid. An increase from room temperature to body temperature showed excellent temperature assisted passivity and nobler surface layer of the high entropy alloy, resulting in four times better wear resistance compared to stainless steel. Stem cells and osteoblast cells displayed proliferation and migratory behavior, indicating in vitro biocompatibility. Several filopodia extensions on the cell periphery indicated early osteogenic commitment, and cell adhesion on the high entropy alloy. These results pave the way for utilizing the unique combination of tribo-corrosion resistance, excellent mechanical properties, and biocompatibility of MoNbTaTiZr high entropy alloy to develop bioimplants with improved service life and lower risk of implant induced cytotoxicity in the host body.

Intron-specific Single Nucleotide Polymorphisms of Fat Mass and Obesity- Associated Gene in Obese and Overweight Individuals of the Indian Adult Population- A Pilot Study.

BACKGROUND: The Fat mass and obesity-associated gene (FTO) and its involvement in weight gain and obesity is well-known. However, no reports have been published on the Indian population regarding the relationship between single nucleotide polymorphisms (SNPs) in its intronic region and obesity. The aim of this pilot study was to evaluate the frequency and association of SNPs in intron-1 of the FTO gene in obese and overweight Indian adults. METHODS: This study group consisted of 80 adults, aged 23.5 ± 8.9 yr, with a mean BMI of 28.8 ± 6.2 kg/m2. Genomic DNA was isolated, exons1-3 & intron1 of FTO were amplified using polymerase chain reaction and sequenced by ABI sequencing detection system. The reported SNPs rs1420185, rs8050136, rs1121980 and rs55872725 were checked for their presence or absence in this group of the adult Indian population. RESULTS: No mutations were found in the exonic sequence of FTO, however, the association of rs1420185, rs8050136, rs1121980 and rs55872725 SNPs was identified in this population. The genotypic frequency at FTO rs8050136 was 32.2% for C>A, at rs55872725 it was 45.7% for C>T, at rs1420185 it was 27.1% for T>C and at rs1121980 it was 30.5% for G>A. All four SNPs in combination were observed in 6 participants (10.2%), all of whom were found to be either obese or overweight. CONCLUSION: These findings indicate that Indians with these SNPs are most likely to be at increased risk of obesity.

Tetraphenylethylene conjugated p-hydroxyphenacyl: fluorescent organic nanoparticles for the release of hydrogen sulfide under visible light with real-time cellular imaging.

Hydrogen sulfide (H2S) behaves like a two-edged sword, at low concentrations it has beneficial and cytoprotective effects, while at higher concentrations it exhibits toxicity. Hence there is a keen interest in developing light responsive H2S donors with a spatio-temporal controlled release. Herein, we report visible light activatable tetraphenylethylene conjugated p-hydroxyphenacyl (TPE-pHP-H2S) nanoparticles for the release of hydrogen sulfide (H2S) with a real time monitoring ability. Our newly designed photoresponsive single component organic nanoparticle based H2S donor is built by integrating the tetraphenylethylene (TPE) moiety and p-hydroxyphenacyl (pHP) group so that it can display both aggregation-induced emission (AIE) and excited state intramolecular proton transfer (ESIPT) properties. Aggregation-induced emission enhancement was exhibited by our TPE-pHP-H2S NP donor, which was then explored for the cellular imaging application. The ESIPT by the pHP moiety provided unique advantages to our TPE-pHP-H2S NP donor which include (i) the excitation wavelength extended to >410 nm (ii) a large Stokes shift (iii) a low inner filter effect and (iv) real-time monitoring of H2S release by a simple fluorescent colour change. In vitro studies showed that the TPE-pHP-H2S NP donor presents excellent properties like real-time monitoring, photoregulated H2S release and biocompatibility.

NOTCH Signaling Is Essential for Maturation, Self-Renewal, and Tri-Differentiation of In Vitro Derived Human Neural Stem Cells.

Although neural stem cells (NSCs) have potential applications in treating neurological disorders, much still needs to be understood about the differentiation biology for their successful clinical translation. In this study, we aimed at deriving NSCs from human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) and explored the role of Notch signaling in the differentiation process. The hUCB-MSCs were characterized as per guidelines of the International Society of Cellular Therapy. NSCs were successfully generated from hUCB-MSCs by using epidermal and fibroblast growth factors under serum-free conditions. The expression of NSC markers (Nestin and Musashi-1) in the neurospheres generated from hUCB-MSCs in the presence or absence of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT; Notch inhibitor) was immuno-phenotypically characterized by using immunofluorescence. DAPT showed significant (*p < 0.05) downregulated expression of the NSC markers-Nestin and SOX2-at different time points (6 hours, 12 hours, 24 hours, 36 hours, and 5 days) post-treatment. In addition, Mushashi-1 (NSC marker) expression in NSCs was also inhibited after DAPT treatment, which signifies that the process is Notch dependent. These data were further correlated with formation of a reduced average number of neurospheres derived from hUCB-MSCs (2 colonies vs. 11 colonies/field of view) in the presence of DAPT compared with the control (without DAPT). The expression of Notch target genes in NSC cultures (Notch intracellular domain [NICD], HES1, and HES5) was also significantly downregulated after DAPT treatment. In the presence of DAPT, the markers for neuronal (MAP2, NEFH); and glial (GFAP, GLUL, and MBP) lineages were significantly downregulated as seen via immunofluorescence and quantitative polymerase chain reaction, indicating the role of Notch in the tri-differentiation mechanism of NSCs as well. In addition, Notch signaling inhibition induced higher cell death during the lineage commitment of NSCs as measured 3 days (16.9% vs. 8.9%) and 6 days (42.9% vs. 20.8%) postinduction. These results suggest that the efficient derivation of NSCs and their subsequent lineage commitment from hUCB-MSCs requires the Notch signaling pathway.

DADLE enhances viability and anti-inflammatory effect of human MSCs subjected to 'serum free' apoptotic condition in part via the DOR/PI3K/AKT pathway.

AIM: Nutritional deprivation and inflammation-rich zones are the major causative reasons for poor survivability of transplanted mesenchymal stem cells (MSCs). Therefore in the present study, we demonstrated the cytoprotective and anti-inflammatory effects of activated delta (δ)-opioid receptor (DOR) with synthetic peptide [D-Ala2, D-Leu5]-enkephalin (DADLE) treatment on human MSCs cultured in serum-starved condition. MAIN METHODS: Cell viability was measured using MTT and Annexin V/PI assays. Expressions of pro-apoptotic (Bcl2) and anti-apoptotic genes (Bax/Bad), levels of activated p44/42 MAPK, Akt, PI3-kinase-p110γ and cleaved caspase-3 were determined by qPCR and western blot. Levels of secreted cytokines were measured by ELISA. KEY FINDINGS: In comparison to the control, DADLE significantly increased cell survivability under serum deprived condition as confirmed by MTT (71% vs 45%) and Annexin V/PI assays (25.9% vs 3.7%). Significant up-regulation of pro-apoptotic Bcl2 (~2.1 folds), down-regulations of anti-apoptotic Bax/Bad (~2.6/2.7 folds) as well as of cleaved caspase-3, increased expression of PI3kinase subunit p110γ and activation of Akt (Ser473) were observed following DADLE treatment in cells under 'serum deprivation' stress. In addition, DADLE treated hMSCs secreted increased levels of anti-inflammatory cytokines (IL10/IL4/TGF-ß) under serum deprived condition. LPS stimulated macrophages showed abated release of pro-inflammatory cytokines (IL1/TNFα/IL6) when grown in hMSC conditioned 'serum deprived' media treated with DADLE. Both the cytoprotective and anti-inflammatory effects of DADLE were inhibited by the DOR specific antagonist naltrindole. SIGNIFICANCE: The DOR signaling pathway improved cell viability and enhanced anti-inflammatory effect of hMSCs subjected to 'serum deprivation' stress that could have potential therapeutic benefits in reparative medicine.