Monitoring of freezing patterns within 3D collagen-hydroxyapatite scaffolds using infrared thermography.

The importance of cryopreservation in tissue engineering is unceasingly increasing. Preparation, cryopreservation, and storage of tissue-engineered constructs (TECs) at an on-site location offer a convenient way for their clinical application and commercialization. Partial freezing initiated at high sub-zero temperatures using ice-nucleating agents (INAs) has recently been applied in organ cryopreservation. It is anticipated that this freezing technique may be efficient for the preservation of both scaffold mechanical properties and cell viability of TECs. Infrared thermography is an instrumental method to monitor INAs-mediated freezing of various biological entities. In this paper, porous collagen-hydroxyapatite (collagen-HAP) scaffolds were fabricated and characterized as model TECs, whereas infrared thermography was proposed as a method for monitoring the crystallization-related events on their partial freezing down to -25 °C. Intra- and interscaffold latent heat transmission were descriptively evaluated. Nucleation, freezing points as well as the degree of supercooling and duration of crystallization were calculated based on inspection of respective thermographic curves. Special consideration was given to the cryoprotective agent (CPA) composition (Snomax®, crude leaf homogenate (CLH) from Hippophae rhamnoides, dimethyl sulfoxide (Me2SO) and recombinant type-III antifreeze protein (AFP)) and freezing conditions ('in air' or 'in bulk CPA'). For CPAs without ice nucleation activity, thermographic measurements demonstrated that the supercooling was significantly milder in the case of scaffolds present in a CPA solution compared to that without them. This parameter (ΔT, °C) altered with the following tendency: 10 Me2SO (2.90 ± 0.54 ('scaffold in a bulk CPA') vs. 7.71 ± 0.43 ('bulk CPA', P < 0.0001)) and recombinant type-III AFP, 0.5 mg/ml (2.65 ± 0.59 ('scaffold in a bulk CPA') vs. 7.68 ± 0.34 ('bulk CPA', P < 0.0001)). At the same time, in CPA solutions with ice nucleation activity the least degree of supercooling and the longest crystallization duration (Δt, min) for scaffolds frozen 'in air' were documented for CLH from Hippophae rhamnoides (1.57 ± 0.37 °C and 21.86 ± 2.93 min) compared to Snomax, 5 µg/ml (2.14 ± 0.33 °C and 19.91 ± 4.72 min), respectively). Moreover, when frozen 'in air' in CLH from Hippophae rhamnoides, collagen-HAP scaffolds were shown to have the longest ice-liquid equilibrium phase during crystallization and the lowest degree of supercooling followed by alginate core-shell capsules and nanofibrous electrospun fiber mats made of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) (PCL/PLA) blend. The paper offers evidence that infrared thermography provides insightful information for monitoring partial freezing events in TECs when using different freezing containers, CPAs and conditions. This may further TEC-specific cryopreservation with enhanced batch homogeneity and optimization of CPA compositions of natural origin active at warm sub-zero temperatures.

ATP synthase inhibitory factor subunit 1 regulates islet ß-cell function via repression of mitochondrial homeostasis.

Mitochondrial homeostasis is crucial for the function of pancreatic ß-cells. ATP synthase inhibitory factor subunit 1 (IF1) is a mitochondrial protein interacting with ATP synthase to inhibit its enzyme activity. IF1 may also play a role in maintaining ATP synthase oligomerization and mitochondrial inner membrane formation. A recent study confirmed IF1 expresses in ß-cells. IF1 knockdown in cultured INS-1E ß-cells enhances glucose-induced insulin release. However, the role of IF1 in islet ß-cells remains little known. The present study investigates islets freshly isolated from mouse lines with global IF1 knockout (IF1-/-) and overexpression (OE). The glucose-stimulated insulin secretion was increased in islets from IF1-/- mice but decreased in islets from IF1 OE mice. Transmitted Electronic Microscopic assessment of isolated islets revealed that the number of matured insulin granules (with dense core) was relatively higher in IF1-/-, but fewer in IF1 OE islets than those of controlled islets. The mitochondrial ultrastructure within ß-cells of IF1 overexpressed islets was comparable with those of wild-type mice, whereas those in IF1-/- ß-cells showed increased mitochondrial mass. Mitochondrial network analysis in cultured INS-1 ß-cells showed a similar pattern with an increased mitochondrial network in IF1 knockdown cells. IF1 overexpressed INS-1 ß-cells showed a compromised rate of mitochondrial oxidative phosphorylation with attenuated cellular ATP content. In contrast, INS-1 cells with IF1 knockdown showed markedly increased cellular respiration with improved ATP production. These results support that IF1 is a negative regulator of insulin production and secretion via inhibiting mitochondrial mass and respiration in ß-cells. Therefore, inhibiting IF1 to improve ß-cell function in patients can be a novel therapeutic strategy to treat diabetes.

Nischarin Deletion Reduces Oxidative Metabolism and Overall ATP: A Study Using a Novel NISCHΔ5-6 Knockout Mouse Model.

Nischarin (Nisch) is a cytosolic scaffolding protein that harbors tumor-suppressor-like characteristics. Previous studies have shown that Nisch functions as a scaffolding protein and regulates multiple biological activities. In the current study, we prepared a complete Nisch knockout model, for the first time, by deletion of exons 5 and 6. This knockout model was confirmed by Qrt-PCR and Western blotting with products from mouse embryonic fibroblast (MEF) cells. Embryos and adult mice of knockouts are significantly smaller than their wild-type counterparts. Deletion of Nisch enhanced cell migration, as demonstrated by wound type and transwell migration assays. Since the animals were small in size, we investigated Nisch's effect on metabolism by conducting several assays using the Seahorse analyzer system. These data indicate that Nisch null cells have lower oxygen consumption rates, lower ATP production, and lower levels of proton leak. We examined the expression of 15 genes involved in lipid and fat metabolism, as well as cell growth, and noted a significant increase in expression for many genes in Nischarin null animals. In summary, our results show that Nischarin plays an important physiological role in metabolic homeostasis.

Effect of 'in air' freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds.

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen 'in air' and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of 'ready-to-use' TECs.

Diabetic Pregnancy and Maternal High-Fat Diet Impair Mitochondrial Dynamism in the Developing Fetal Rat Heart by Sex-Specific Mechanisms.

Infants born to diabetic or obese mothers are at greater risk of heart disease at birth and throughout life, but prevention is hindered because underlying mechanisms remain poorly understood. Using a rat model, we showed that prenatal exposure to maternal diabetes and a high-fat diet caused diastolic and systolic dysfunction, myocardial lipid accumulation, decreased respiratory capacity, and oxidative stress in newborn offspring hearts. This study aimed to determine whether mitochondrial dynamism played a role. Using confocal live-cell imaging, we examined mitochondrial dynamics in neonatal rat cardiomyocytes (NRCM) from four prenatally exposed groups: controls, diabetes, high-fat diet, and combination exposed. Cardiac expression of dynamism-related genes and proteins were compared, and gender-specific differences were evaluated. Findings show that normal NRCM have highly dynamic mitochondria with a well-balanced number of fusion and fission events. Prenatal exposure to diabetes or a high-fat diet impaired dynamism resulting in shorter, wider mitochondria. Mechanisms of impaired dynamism were gender-specific and protein regulated. Females had higher expression of fusion proteins which may confer a cardioprotective effect. Prenatally exposed male hearts had post-translational modifications known to impair dynamism and influence mitophagy-mediated cell death. This study identifies mitochondrial fusion and fission proteins as targetable, pathogenic regulators of heart health in offspring exposed to excess circulating maternal fuels.

Multipotent stromal cells derived from common marmoset Callithrix jacchus within alginate 3D environment: Effect of cryopreservation procedures.

Multipotent stromal cells derived from the common marmoset monkey Callithrix jacchus (cjMSCs) possess high phylogenetic similarity to humans, with a great potential for preclinical studies in the field of regenerative medicine. Safe and effective long-term storage of cells is of great significance to clinical and research applications. Encapsulation of such cell types within alginate beads that can mimic an extra-cellular matrix and provide a supportive environment for cells during cryopreservation, has several advantages over freezing of cells in suspension. In this study we have analysed the effect of dimethyl sulfoxide (Me2SO, 2.5-10%, v/v) and pre-freeze loading time of alginate encapsulated cjMSCs in Me2SO (0-45 min) on the viability and metabolic activity of the cells after freezing using a slow cooling rate (-1°C/min). It was found that these parameters affect the stability and homogeneity of alginate beads after thawing. Moreover, the cjMSCs can be frozen in alginate beads with lower Me2SO concentration of 7.5% after 30 min of loading, while retaining high cryopreservation outcome. We demonstrated the maximum viability, membrane integrity and metabolic activity of the cells under optimized, less cytotoxic conditions. The results of this study are another step forward towards the application of cryopreservation for the long-term storage and subsequent applications of transplants in cell-based therapies.

IF1 Promotes Cellular Proliferation and Inhibits Oxidative Phosphorylation in Mouse Embryonic Fibroblasts under Normoxia and Hypoxia.

ATP synthase inhibitory factor subunit 1 (IF1) is an inhibitory subunit of mitochondrial ATP synthase, playing a crucial role in regulating mitochondrial respiration and energetics. It is well-established that IF1 interacts with the F1 sector of ATP synthase to inhibit the reversal rotation and, thus, ATP hydrolysis. Recent evidence supports that IF1 also inhibits forward rotation or the ATP synthesis activity. Adding to the complexity, IF1 may also facilitate mitophagy and cristae formation. The implications of these complex actions of IF1 for cellular function remain obscure. In the present study, we found that IF1 expression was markedly upregulated in hypoxic MEFs relative to normoxic MEFs. We investigate how IF1 affects cellular growth and function in cultured mouse embryonic fibroblasts derived from mouse lines with systemic IF1 overexpression and knockout under normoxia and hypoxia. Cell survival and proliferation analyses revealed that IF1 overexpression exerted limited effects on cellular viability but substantially increased proliferation under normoxia, whereas it facilitated both cellular viability and proliferation under hypoxia. The absence of IF1 may have a pro-survival effect but not a proliferative one in both normoxia and hypoxia. Cellular bioenergetic analyses revealed that IF1 suppressed cellular respiration when subjected to normoxia and was even more pronounced when subjected to hypoxia with increased mitochondrial ATP production. In contrast, IF1 knockout MEFs showed markedly increased cellular respiration under both normoxia and hypoxia with little change in mitochondrial ATP. Glycolytic stress assay revealed that IF1 overexpression modestly increased glycolysis in normoxia and hypoxia. Interestingly, the absence of IF1 in MEFs led to substantial increases in glycolysis. Therefore, we conclude that IF1 mainly inhibits cellular respiration and enhances cellular glycolysis to preserve mitochondrial ATP. On the other hand, IF1 deletion can significantly facilitate cellular respiration and glycolysis without leading to mitochondrial ATP deficit.

Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells.

Adenosine 5' triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease.

Multiomics Approach Reveals an Important Role of BNIP3 in Myocardial Remodeling and the Pathogenesis of Heart Failure with Reduced Ejection Fraction.

Previous work showed a role of BNIP3 in myocardial remodeling and progression to HFrEF. We utilized a multiomics approach to unravel BNIP3-related molecular mechanisms in the pathogenesis of HFrEF. BNIP3 knockdown in HFrEF improved glycolysis, pyruvate metabolism, branched-chain amino acid catabolism, and oxidative phosphorylation, and restored endoplasmic reticulum (ER)-mitochondrial (mt) calcium and ion homeostasis. These effects of BNIP3 on cardiac metabolism were related to its interaction and downregulation, and/or phosphorylation, of specific mt-proteins involved in the aforementioned metabolic pathways, including the MICOS and SLC25A families of carrier proteins. BNIP3 affected ER-mt-calcium and ion homeostasis via its interaction-induced VDAC1 dimerization and modulation of VDAC1 phosphorylation at Ser104 and Ser241, and the downregulation of LETM1. At the ER level, BNIP3 interacted with the enzyme SERCA2a and the PKA signaling complex, leading to the downregulation of SERCA2a and PKA-mediated Ser16 phospholamban phosphorylation. Additionally, BNIP3 attenuated AMPK and PRKCE activity by modulating AMPK phosphorylation at Ser485/491 and Ser377 residues, and PRKCE phosphorylation at Thr521 and Thr710 residues. BNIP3 also interacted with sarcomeric, cytoskeletal, and cellular transcription and translation proteins, and affected their expression and/or phosphorylation. In conclusion, BNIP3 modulates multiple pathobiological processes and constitutes an attractive therapeutic target in HFrEF.

A combination of novel NSC small molecule inhibitor along with doxorubicin inhibits proliferation of triple-negative breast cancer through metabolic reprogramming.

Treatment of patients with triple-negative breast cancer (TNBC) has been challenging due to the absence of well-defined molecular targets and the highly invasive and proliferative nature of TNBC cells. Current treatments against TNBC have shown little promise due to high recurrence rate in patients. Consequently, there is a pressing need for novel and efficacious therapies against TNBC. Here, we report the discovery of a novel small molecule inhibitor (NSC33353) with potent anti-tumor activity against TNBC cells. The anti-proliferative effects of this small molecule inhibitor were determined using 2D and 3D cell proliferation assays. We found that NSC33353 significantly reduces the proliferation of TNBC cells in these assays. Using proteomics, next generation sequencing (NGS), and gene enrichment analysis, we investigated global regulatory pathways affected by this compound in TNBC cells. Proteomics data indicate a significant metabolic reprograming affecting both glycolytic enzymes and energy generation through oxidative phosphorylation. Subsequently, using metabolic (Seahorse) and enzymatic assays, we validated our proteomics and NGS analysis findings. Finally, we showed the inhibitory and anti-tumor effects of this small molecule in vitro and confirmed its inhibitory activity in vivo. Doxorubicin is one of the most effective agents in the treatment of TNBC and resistance to this drug has been a major problem. We show that the combination of NSC33353 and doxorubicin suppresses the growth of TNBC cells synergistically, suggesting that NSC33353 enhances TNBC sensitivity to doxorubicin. In summary, our data indicate that the small molecule inhibitor, NSC33353, exhibits anti-tumor activity in TNBC cells, and works in a synergistic fashion with a well-known chemotherapeutic agent.

Improved in vitro models for preclinical drug and formulation screening focusing on 2D and 3D skin and cornea constructs.

The present overview deals with current approaches for the improvement of in vitro models for preclinical drug and formulation screening which were elaborated in a joint project at the Center of Pharmaceutical Engineering of the TU Braunschweig. Within this project a special focus was laid on the enhancement of skin and cornea models. For this reason, first, a computation-based approach for in silico modeling of dermal cell proliferation and differentiation was developed. The simulation should for example enhance the understanding of the performed 2D in vitro tests on the antiproliferative effect of hyperforin. A second approach aimed at establishing in vivo-like dynamic conditions in in vitro drug absorption studies in contrast to the commonly used static conditions. The reported Dynamic Micro Tissue Engineering System (DynaMiTES) combines the advantages of in vitro cell culture models and microfluidic systems for the emulation of dynamic drug absorption at different physiological barriers and, later, for the investigation of dynamic culture conditions. Finally, cryopreserved shipping was investigated for a human hemicornea construct. As the implementation of a tissue-engineering laboratory is time-consuming and cost-intensive, commercial availability of advanced 3D human tissue is preferred from a variety of companies. However, for shipping purposes cryopreservation is a challenge to maintain the same quality and performance of the tissue in the laboratory of both, the provider and the customer.

Novel chitin scaffolds derived from marine sponge Ianthella basta for tissue engineering approaches based on human mesenchymal stromal cells: Biocompatibility and cryopreservation.

The extraordinary biocompatibility and mechanical properties of chitinous scaffolds from marine sponges endows these structures with unique properties that render them ideal for diverse biomedical applications. In the present work, a technological route to produce "ready-to-use" tissue-engineered products based on poriferan chitin is comprehensively investigated for the first time. Three key stages included isolation of scaffolds from the marine demosponge Ianthella basta, confirmation of their biocompatibility with human mesenchymal stromal cells, and cryopreservation of the tissue-like structures grown within these scaffolds using a slow cooling protocol. Biocompatibility of the macroporous, flat chitin scaffolds has been confirmed by cell attachment, high cell viability and the ability to differentiate into the adipogenic lineage. The viability of cells cryopreserved on chitin scaffolds was reduced by about 30% as compared to cells cryopreserved in suspension. However, the surviving cells were able to retain their differentiation potential; and this is demonstrated for the adipogenic lineage. The results suggest that chitin from the marine demosponge I. basta is a promising, highly biocompatible biomaterial for stem cell-based tissue-engineering applications.

Xeno-Free Cryopreservation of Bone Marrow-Derived Multipotent Stromal Cells from Callithrix jacchus.

In the previous decade, numerous biobanks were established and have created large markets for the storage of bioactive compounds, cells, and tissues for medical and diagnostic applications. For in vivo clinical and therapeutic purposes, it is critical to use well-defined and xeno-free components during cultivation, preservation, and transplantation of biological material. Safe and efficacious storage of bioactive molecules, cells, and tissues, without the addition of undefined medium components, minimizes risks of zoonotic disease transmission and is thus an essential and desirable prerequisite for biobanks. This gives rise to a need for well-characterized and serum-free freezing media for application in cryopreservation. For this purpose, cryobiological additives such as methylcellulose, poloxamer-188, and α-tocopherol, which have previously been shown to exhibit a cytoprotective activity, have been investigated for cryoprotection on stem cells. With this strategy, the application of fetal bovine serum (FBS) could be avoided and the concentration of toxic cryoprotective agents such as dimethyl sulfoxide (DMSO) could be reduced. Our results suggest that the viability, as well as the adipogenic and osteogenic differentiation capacity of the thawed bone marrow-derived multipotent stromal stem cells, could be maintained using a freezing medium without FBS consisting of methylcellulose, poloxamer, and α-tocopherol with only 2.5% DMSO (% v/v).

Porous biomorphic silicon carbide ceramics coated with hydroxyapatite as prospective materials for bone implants.

Porous and cytocompatible silicon carbide (SiC) ceramics derived from wood precursors and coated with bioactive hydroxyapatite (HA) and HA-zirconium dioxide (HA/ZrO2) composite are materials with promising application in engineering of bone implants due to their excellent mechanical and structural properties. Biomorphic SiC ceramics have been synthesized from wood (Hornbeam, Sapele, Tilia and Pear) using a forced impregnation method. The SiC ceramics have been coated with bioactive HA and HA/ZrO2 using effective gas detonation deposition approach (GDD). The surface morphology and cytotoxicity of SiC ceramics as well as phase composition and crystallinity of deposited coatings were analyzed. It has been shown that the porosity and pore size of SiC ceramics depend on initial wood source. The XRD and FTIR studies revealed the preservation of crystal structure and phase composition of in the HA coating, while addition of ZrO2 to the initial HA powder resulted in significant decomposition of the final HA/ZrO2 coating and formation of other calcium phosphate phases. In turn, NIH 3T3 cells cultured in medium exposed to coated and uncoated SiC ceramics showed high re-cultivation efficiency as well as metabolic activity. The recultivation efficiency of cells was the highest for HA-coated ceramics, whereas HA/ZrO2 coating improved the recultivation efficiency of cells as compared to uncoated SiC ceramics. The GDD method allowed generating homogeneous HA coatings with no change in calcium to phosphorus ratio. In summary, porous and cytocompatible bio-SiC ceramics with bioactive coatings show a great promise in construction of light, robust, inexpensive and patient-specific bone implants for clinical application.