Photoacoustic processing of decellularized extracellular matrix for biofabricating living constructs.

The diverse biomolecular landscape of tissue-specific decellularized extracellular matrix (dECM) biomaterials provides a multiplicity of bioinstructive cues to target cells, rendering them highly valuable for various biomedical applications. However, the isolation of dECM biomaterials entails cumbersome xenogeneic enzymatic digestions and also additional inactivation procedures. Such, increases processing time, increments costs and introduces residues of non-naturally present proteins in dECM formulations that remain present even after inactivation. To overcome these limitations, herein we report an innovative conjugation of light and ultrasound-mediated dECM biomaterial processing for fabricating dECM biomaterials. Such approach gathers on ultrasound waves to facilitate dECM-in-liquid processing and visible light photocrosslinking of tyrosine residues naturally present in dECM biomaterials. This dual step methodology unlocked the in-air production of cell laden dECM hydrogels or programmable dECM hydrogel spherical-like beads by using superhydrophobic surfaces. These in-air produced units do not require any additional solvents and successfully supported both fibroblasts and breast cancer cells viability upon encapsulation or surface seeding. In addition, the optimized photoacoustic methodology also enabled a rapid formulation of dECM biomaterial inks with suitable features for biofabricating volumetrically defined living constructs through embedded 3D bioprinting. The biofabricated dECM hydrogel constructs supported cell adhesion, spreading and viability for 7 days. Overall, the implemented photoacoustic processing methodology of dECM biomaterials offers a rapid and universal strategy for upgrading their processing from virtually any tissue. STATEMENT OF SIGNIFICANCE: Leveraging decellularized extracellular matrix (dECM) as cell instructive biomaterials has potential to open new avenues for tissue engineering and in vitro disease modelling. The processing of dECM remains however, lengthy, costly and introduces non-naturally present proteins in the final biomaterials formulations. In this regard, here we report an innovative light and ultrasound two-step methodology that enables rapid dECM-in-liquid processing and downstream photocrosslinking of dECM hydrogel beads and 3D bioprinted constructs. Such photoacoustic based processing constitutes a universally applicable method for processing any type of tissue-derived dECM biomaterials.

Brewer's Spent Yeast Cell Wall Polysaccharides as Vegan and Clean Label Additives for Mayonnaise Formulation.

Brewer's spent yeast (BSY) mannoproteins have been reported to possess thickening and emulsifying properties. The commercial interest in yeast mannoproteins might be boosted considering the consolidation of their properties supported by structure/function relationships. This work aimed to attest the use of extracted BSY mannoproteins as a clean label and vegan source of ingredients for the replacement of food additives and protein from animal sources. To achieve this, structure/function relationships were performed by isolating polysaccharides with distinct structural features from BSY, either by using alkaline extraction (mild treatment) or subcritical water extraction (SWE) using microwave technology (hard treatment), and assessment of their emulsifying properties. Alkaline extractions solubilized mostly highly branched mannoproteins (N-linked type; 75%) and glycogen (25%), while SWE solubilized mannoproteins with short mannan chains (O-linked type; 55%) and (1→4)- and (ß1→3)-linked glucans, 33 and 12%, respectively. Extracts with high protein content yielded the most stable emulsions obtained by hand shaking, while the extracts composed of short chain mannans and ß-glucans yielded the best emulsions by using ultraturrax stirring. ß-Glucans and O-linked mannoproteins were found to contribute to emulsion stability by preventing Ostwald ripening. When applied in mayonnaise model emulsions, BSY extracts presented higher stability and yet similar texture properties as the reference emulsifiers. When used in a mayonnaise formulation, the BSY extracts were also able to replace egg yolk and modified starch (E1422) at 1/3 of their concentration. This shows that BSY alkali soluble mannoproteins and subcritical water extracted ß-glucans can be used as replacers of animal protein and additives in sauces.

Advances in bioengineering pancreatic tumor-stroma physiomimetic Biomodels.

Pancreatic cancer exhibits a unique bioarchitecture and desmoplastic cancer-stoma interplay that governs disease progression, multi-resistance, and metastasis. Emulating the biological features and microenvironment heterogeneity of pancreatic cancer stroma in vitro is remarkably complex, yet highly desirable for advancing the discovery of innovative therapeutics. Diverse bioengineering approaches exploiting patient-derived organoids, cancer-on-a-chip platforms, and 3D bioprinted living constructs have been rapidly emerging in an endeavor to seamlessly recapitulate major tumor-stroma biodynamic interactions in a preclinical setting. Gathering on this, herein we showcase and discuss the most recent advances in bio-assembling pancreatic tumor-stroma models that mimic key disease hallmarks and its desmoplastic biosignature. A reverse engineering perspective of pancreatic tumor-stroma key elementary units is also provided and complemented by a detailed description of biodesign guidelines that are to be considered for improving 3D models physiomimetic features. This overview provides valuable examples and starting guidelines for researchers envisioning to engineer and characterize stroma-rich biomimetic tumor models. All in all, leveraging advanced bioengineering tools for capturing stromal heterogeneity and dynamics, opens new avenues toward generating more predictive and patient-personalized organotypic 3D in vitro platforms for screening transformative therapeutics targeting the tumor-stroma interplay.

Organotypic 3D decellularized matrix tumor spheroids for high-throughput drug screening.

Decellularized extracellular matrix (dECM) is emerging as a valuable tool for generating 3D in vitro tumor models that better recapitulate tumor-stroma interactions. However, the development of dECM-3D heterotypic microtumors exhibiting a controlled morphology is yet to be materialized. Precisely controlling microtumors morphologic features is key to avoid an inaccurate evaluation of therapeutics performance during preclinical screening. To address this, herein we employed ultra-low adhesion surfaces for bioengineering organotypic 3D metastatic breast cancer-fibroblast models enriched with dECM microfibrillar fragments, as a bottom-up strategy to include major matrix components and their associated biomolecular cues during the early stages of 3D microtissue spheroids assembly, simulating pre-existing ECM presence in the in vivo setting. This biomimetic approach enabled the self-assembly of dECM-3D tumor-stroma spheroids with tunable size and reproducible morphology. Along time, dECM enriched and stroma-rich microtumors exhibited necrotic core formation, secretion of key biomarkers and higher cancer-cell specific resistance to different chemotherapeutics in comparison to standard spheroids. Exometabolomics profiling of dECM-Spheroid in vitro models further identified important breast cancer metabolic features including glucose/pyruvate consumption and lactate excretion, which suggest an intense glycolytic activity, recapitulating major hallmarks of the native microenvironment. Such organotypic dECM-enriched microtumors overcome the morphologic variability generally associated with cell-laden dECM models, while providing a scalable testing platform that can be foreseeable leveraged for high-throughput screening of candidate therapeutics.

Consistent Inclusion of Mesenchymal Stem Cells into In Vitro Tumor Models.

Over recent years, the role of distinct mesenchymal stem cell populations in cancer progression has become increasingly evident. In this regard, developing in vitro preclinical tumor models capable of portraying tumor-associated mesenchymal stem cells (TA-MSCs) interactions with the tumor microenvironment (TME), cellular and extracellular components, would allow to improve the predictive potential of these platforms and expedite preclinical drug screening. Although recent studies successfully developed in vitro tumor models in which the biomolecular and cellular behaviors of TA-MSCs were recapitulated in the context of their interactions with specific TME components, no consensus has yet been reached regarding distinct TA-MSCs influence in the evolution of solid tumors. The paradoxical observations regarding the roles of MSCs on in vitro tumor models can in part be associated to a lack of standardization in how MSCs integration is performed. Herein, we summarize some of the main parameters linked to phenotypic variations established upon MSCs inclusion and interaction within in vitro tumor models. A critical overview of recent studies and how standardization of key parameters could improve the reproducibility and predictability of current preclinical validation models containing MSCs is also provided.

Bioimaging of Mesenchymal Stem Cells Spatial Distribution and Interactions with 3D In Vitro Tumor Spheroids.

In solid tumors, mesenchymal stem cells (MSCs) are recognized to establish complex intercommunication networks with cancer cells and to significantly influence their invasion and metastasis potential. Such bidirectional interplay occurs between both tissue resident/tumor-associated MSCs (TA-MSCs) and also tumor infiltrating MSCs (TM-MSCs) that migrate from distant sites such as the bone marrow. Interestingly, malignant cells interactions with MSCs in the tumor microenvironment extends beyond conventional exchanges of signaling factors and extracellular vesicles, including unconventional direct exchanges of intracellular components, or cancer cells cannibalism of MSCs. In the context of 3D in vitro tumor models, cell tracking assays making use of cell-labeling probes such as membrane penetrating dyes, can be leveraged to shed light on these events, and allow researchers to analyze overtime cell-to-cell spatial distribution, fusion, internal organization, and changes in co-cultured populations ratios. Herein, we describe a high-throughput compatible method through which MSCs positioning and permanence within in vitro 3D multicellular tumor spheroid models (3D-MCTS) can be tracked overtime. Although we have focused on the interactions of human bone marrow-derived MSCs (hBM-MSCs) within heterotypic lung cancer A549 3D-MCTS, these procedures can be implemented for other 3D tumor spheroid models and types of cells, taking into consideration that optimization steps are undertaken.

Screening of dual chemo-photothermal cellular nanotherapies in organotypic breast cancer 3D spheroids.

Living therapeutics approaches that exploit mesenchymal stem cells (MSCs) as nanomedicine carriers are highly attractive due to MSCs native tropism toward the 3D tumor microenvironment. However, a streamlined pre-clinical evaluation of nano-in-cell anti-cancer therapies remains limited by the lack of in vitro testing platforms for screening MSCs-3D microtumor interactions. Herein we generated dense breast cancer mono and heterotypic 3D micro-spheroids for evaluating MSCs-solid tumors interactions and screen advanced nano-in-MSCs therapies. Breast cancer monotypic and heterotypic models comprising cancer cells and cancer associated fibroblasts (CAFs) were self-assembled under controlled conditions using the liquid overlay technique. The resulting microtumors exhibited high compactness, reproducible morphology and necrotic regions, similarly to native solid tumors. For evaluating tumoritropic therapies in organotypic tumor-stroma 3D models, theranostic polydopamine nanoparticles loaded with indocyanine green-doxorubicin combinations (PDA-ICG-DOX) were synthesized and administered to human bone-marrow derived MSCs (hBM-MSCs). The dual-loaded PDA nano-platforms were efficiently internalized, exhibited highly efficient NIR-light responsivity and assured MSCs viability up to 3 days. The administration of PDA-ICG-DOX nano-in-MSC tumoritropic units to microtumor models was performed in ultra-low adhesion surfaces for simulating in vitro the stem cell-tumor interactions observed in the in vivo scenario. Bioimaging analysis revealed hBM-MSCs adhesion to 3D cancer cells mass and MSCs-chemo-photothermal nanotherapeutics exhibited higher anti-tumor potential when compared to their standalone chemotherapy treated 3D tumor counterparts. Overall, the proposed methodology is suitable for evaluating MSCs-microtumors individualized interactions and enables a rapid high-throughput screening of tumoritropic therapies bioperformance.

Decellularized Extracellular Matrix for Bioengineering Physiomimetic 3D in Vitro Tumor Models.

Recent advances in the extraction and purification of decellularized extracellular matrix (dECM) obtained from healthy or malignant tissues open new avenues for engineering physiomimetic 3D in vitro tumor models, which closely recapitulate key biomolecular hallmarks and the dynamic cancer cell-ECM interactions in the tumor microenvironment. We review current and upcoming methodologies for chemical modification of dECM-based biomaterials and advanced bioprocessing into organotypic 3D solid tumor models. A comprehensive review of disruptive advances and shortcomings of exploring dECM-based biomaterials for recapitulating the native tumor-supporting matrix is also provided. We hope to drive the discussion on how 3D dECM testing platforms can be leveraged for generating microphysiological tumor surrogates that generate more robust and predictive data on therapeutic bioperformance.

Hydrogel 3D in vitro tumor models for screening cell aggregation mediated drug response.

Hydrogel-based 3D in vitro models comprising tumor ECM-mimetic biomaterials exhibit superlative potential as preclinical testing platforms for drug discovery and bioperformance screening. However, during hydrogel design and testing stages, the ideal selection between cancer cell laden 3D models or spheroid embedded hydrogel platforms remains to be elucidated. Selecting a disease-mimicking cellular arrangement within ECM hydrogels is paramount for anti-cancer therapeutics performance evaluation and may lead to differential outcomes. To investigate the effects assigned to varying cellular-arrangement, we developed dense 3D spheroid microtumors and cell-laden MG-63 osteosarcoma platforms embedded in GelMA and Matrigel ECM-mimetic scaffolds. These platforms enabled cancer cells/3D microtissues maturation and lorlatinib drug performance screening. Initial 3D spheroids assembly via the liquid overlay technique, resulted in the fabrication of dense cellular aggregates with reproducible size, morphology and necrotic core formation, thus mimicking the native tumor. Upon in vitro maturation, MG-63 spheroids encapsulated in hydrogel scaffolds exhibited significantly higher invasion and drug resistance than their cell laden hydrogel counterparts. Such data reveals inherent physiological and drug response variances among randomly distributed osteosarcoma cells and 3D spheroid-laden hydrogels. Overall, this highlights the importance of evaluating different cellular aggregation states when designing ECM-mimetic hydrogels for in vitro tumor modeling and high-throughput screening of anti-cancer therapeutics.

Mesenchymal Stem Cells Relevance in Multicellular Bioengineered 3D In Vitro Tumor Models.

In vitro 3D tumor microenvironment mimicking models are gathering momentum as alternatives to traditional 2D flat monolayer cultures due to their potential for recapitulating major cancer hallmarks. To fulfill such potential, it is crucial that 3D tumor testing platforms completely emulate in vitro the complex in vivo tumor niche and its cellular constituents. Mesenchymal stem cells (MSCs) are recognized to play a pivotal multi-modulatory role in cancer, generating interest as biological targets and as key tumor suppressing, or tumor promoting effectors. This review discusses the biological influence of different types of MSCs in the tumor microenvironment and showcases recent studies that engineer 3D MSCs-cancer cells co-cultures as advanced in vitro therapy testing platforms. A special focus is given to MSCs-cancer 3D co-culture set-up parameters, challenges, and future opportunities. Understanding cancer-MSCs crosstalk and their underlying effects is envisioned to support the development of advanced 3D in vitro disease models for discovery of forefront cancer treatments.