Human tissue-derived organoids hold strong potential for personalized medicine and cell therapy, but this requires large cell quantities. Conventional organoid culture systems remain labor-intensive, are difficult to scale, and lack process control. Here, we present a novel strategy using an automated bioreactor platform that enables large-scale expansion of human liver organoids. Human liver organoids were expanded for 14 days in a single bioreactor suspension culture bag and compared with spinner flasks and static dome cultures. Cell yield, viability, fold expansion, morphology, and phenotypic markers (LGR5, E-cadherin, Vimentin, Ki67) were assessed. The system’s uninterrupted workflow enabled seamless transition to differentiation: using integrated perfusion, we performed a direct medium switch from expansion to hepatic differentiation without harvesting or disrupting the culture. Commitment to the hepatic lineage was evaluated by expression of ALB, CYP3A4, MRP2, and HNF4A. By day 14, the bioreactor generated an average of 5.63 × 108 (± 1.1 × 108) viable cells, while spinner flasks reached 1.22 × 108 (± 4.26 × 107) cells, while static cultures yielded only 4.02 × 105 (± 2.81 × 105), making the bioreactor’s output 1400 times greater than static cultures (p = 0.022) and nearly five times higher than spinner flasks. This substantial gain in absolute cell yield is a promising indicator for downstream translation. Organoids preserved phenotypic integrity and proliferative capacity as shown by sustained expression of LGR5, E-cadherin, and Ki67. Bioreactor-cultured organoids exhibited robust growth and intact cyst-like morphology with a large size, due to the absence of mechanical fragmentation and related cellular stress. As a proof-of-principle, bioreactor-grown organoids differentiated efficiently toward the hepatic lineage, as evidenced by a downregulated gene expression of LGR5 and Ki67, with elevated gene expression of ALB, CYP3A4, MRP2, and HNF4A, along with an upregulated secretion of Albumin. The system establishes a closed, monitored, and scalable upstream workflow for liver organoid expansion. This work represents a significant step toward organoid production for future cell therapy and regenerative medicine applications, while maintaining phenotypic stability and differentiation capacity.

Drug-induced liver injury (DILI) remains a major cause of acute liver failure and drug withdrawal from the market. Recently developed three-dimensional (3D) hepatic in vitro systems exhibit improved functionality and drug sensitivity compared with conventional two-dimensional cultures. These 3D models range from simple physiologic-like culture systems to advanced bioreactors with dynamic flow to provide sufficient nutrients and consistent drug exposure. However, whether dynamic perfusion improves sensitivity and reproducibility of hepatotoxicity testing remains unclear. Here, we developed a tailor-made perfusion platform to support volumetric bioprinted hepatic constructs for hepatotoxicity testing. The constructs consist of intrahepatic cholangiocyte organoids (ICOs) differentiated towards hepatocyte lineage and embedded in a gelatin methacryloyl bioresin. For toxicity evaluation, the hepatocyte-like ICO constructs were exposed to prolonged subtoxic acetaminophen treatment (10 mM, 7 days). The perfusion system effectively maintained and enhanced hepatocyte differentiation, evidenced by upregulated hepatic markers under perfused conditions compared to static controls. Testing of acetaminophen hepatotoxicity revealed that the perfused constructs displayed elevated cellular injury, with markedly higher liver injury markers relative to controls. Collectively, this study demonstrates the successful application of perfusion-based 3D model culture and highlights its potential as a more physiological platform for hepatotoxicity risk assessment in drug discovery and regenerative medicine.

Human tissue engineering holds great promise for creating physiological models while facing challenges replicating natural complexity, including cellular and extracellular cues. Current approaches often miss the incorporation of major bioengineering factors (i.e., cellular complexity, well-defined extracellular matrix (ECM) mimicry, dynamic stimuli). We bioengineered liver tissues (BLTs) utilizing human intrahepatic cholangiocyte organoids (ICOs), hepatic stellate cells (HSCs), and mesenchymal stromal cells (MSCs), a synthetic polyisocynide (PIC)- based hydrogel, and dynamic suspension culture (DS) to represent major bioengineering factors. Both mesenchymal cells (HSCs and MSCs) accelerated organoid growth and promoted spontaneous complex liver-like microtissue (BLT) formation. DS and PIC further improved either BLT formation or functionality. Transcriptomic analyses revealed the integrated cellular and extracellular cues in BLT formation and maintenance. To conclude, organoids augmented with mesenchymal cells in chemically defined hydrogels yield functional BLTs suitable for disease modelling, drug screening, and toxicity tests, and form an important basis for future development of larger liver tissues for in vivo transplantation. The bioengineering strategy developed in this study can be extended to engineer other types of tissues and also be utilized to investigate the interaction of different bioengineering factors.

Background & Aims: Following liver damage, ductular reaction often coincides with liver fibrosis. Proliferation of hepatic progenitor cells is observed in ductular reaction, whereas activated hepatic stellate cells (HSCs) are the main drivers of liver fibrosis. These observations may suggest a functional interaction between these 2 cell types. Here, we report on an in vitro co-culture system to examine these interactions and validate their co-expression in human liver explants. Methods: In a 3D organoid co-culture system, we combined freshly isolated quiescent mouse HSCs and fluorescently labeled progenitor cells (undifferentiated intrahepatic cholangiocyte organoids), permitting real-time observation of cell morphology and behavior. After 7 days, cells were sorted based on the fluorescent label and analyzed for changes in gene expression. Results: In the 3D co-culture system, the proliferation of progenitor cells is enhanced, and HSCs are activated, recapitulating the cellular events observed in the patient liver. Both effects in 3D co-culture require close contact between the 2 different cell types. HSC activation during 3D co-culture differs from quiescent (3D mono-cultured) HSCs and activated HSCs on plastic (2D mono-culture). Upregulation of a cluster of genes containing Aldh1a2, Cthrc1, and several genes related to frizzled binding/Wnt signaling were exclusively observed in 3D co-cultured HSCs. The localized co-expression of specific genes was confirmed by spatial transcriptomics in human liver explants. Conclusion: An in vitro 3D co-culture system provides evidence for direct interactions between HSCs and progenitor cells, which are sufficient to drive responses that are similar to those seen during ductular reaction and fibrosis. This model paves the way for further research into the cellular basis of liver pathology.

Bioprinting is currently the most promising method to biofabricate complex tissues in vitro with the potential to transform the future of organ transplantation and drug discovery. Efforts to create such tissues are, however, almost exclusively based on animal-derived materials, such as gelatin methacryloyl, which have demonstrated efficacy in bioprinting of complex tissues. While these materials are already used in clinical applications, uncertainty about their safety still remains due to their animal origin. Alternatively, synthetic bioinks have been developed that match the printability of natural bioinks but lack their biological complexity, and thereby often fail to support cell growth and facilitate tissue formation. Additionally, most synthetic materials do not meet the mechanical demands of bioprint stable constructs while providing a suitable environment for cells to grow, limiting the number of available bioinks. To bridge this gap and synergize bioprinting and 3D cell culture, we developed a polyethylene glycol-based bioink system to promote the growth and spreading of cell spheroids that consist of human primary endothelial cells and fibroblasts. The 3D bioprinted centimeter-scale constructs have a high shape fidelity and accelerated softening to provide sufficient space for cells to grow. Adjusting the rate of degradability, induced by the integration of ester-functionalized crosslinkers in addition to protease cleavable crosslinkers into the hydrogel network, improves the growth of spheroids in larger printed hydrogel constructs containing an interconnected channel structure. The perfusable constructs enable extensive spheroid sprouting and the formation of a cellular network upon fusion of sprouts as initial steps toward tissue formation with the potential for clinical translation.

Background: Diabetes mellitus (DM) is a common potentially life-threatening endocrine disorder in pets and humans. Since only symptomatic treatment is available, a more sustainable treatment is urgently needed. Objective: The aim of this study is to establish functional differentiated canine pancreatic β-cells that release insulin upon glucose stimulus. Methods: Pancreatic tissue was obtained from surplus material of healthy dogs (n = 4), euthanized for non-pancreatic related research. Ductal cells were isolated and expanded in dog pancreas expansion media (dpEM) and differentiated and maturated in five sequentially added pancreas differentiation media (PDMs). Gene expression was analyzed by reversed transcriptase qPCR (RT-qPCR), and insulin release was analyzed with a canine-specific ELISA. Results: Canine pancreatic ductal cells (LGR5 and SOX9 expression) were differentiated into β-cells expressing key β-cell-related genes: Pancreatic and duodenal homeobox 1 (PDX1), NK6 Homeobox 1 (NKX6.1), Glucose Transporter Type 2 (GLUT2), Proprotein convertase subtilisin/kexin type 1 (PCSK1), and low levels of insulin. Neither Glucagon (α-cells) nor LGR5 and SOX9 were expressed, and somatostatin was expressed at low levels. The differentiated cells released insulin upon glucose stimulation. Conclusion and implications: The step-by-step differentiation protocol, mimicking pancreatic organogenesis, resulted in β-cells secreting insulin levels suitable for β-cell disease modelling. It remains to be seen if stem cells from diseased animals behave similarly.

Novel treatments using autologous or allogeneic mesenchymal stromal cells (MSC) are gaining traction in both human and veterinary medicine. MSC secrete multiple factors with immunomodulatory properties, making them promising for treating immune-mediated and inflammatory conditions. However, their application is challenged by donor variability, inconsistent tissue sources and lack of standardized manufacturing protocols, especially in veterinary medicine approaches exploiting the use of canine and feline MSC. Adipose tissue (AT), in particular, is a readily accessible MSC source for both species. This study applied a previously established enzymatic isolation method and focused on developing an efficient ex vivo expansion protocol for canine and feline adipose tissue-derived MSC, emphasizing the selection of a cost-effective culture medium that ensures high viability and proliferation. Subcutaneous AT was collected from 5 female dogs and 5 female cats during ovariohysterectomy. Samples were processed enzymatically, and isolated cells were cultured in basal medium supplemented with different concentrations of fetal bovine serum (FBS) (either standard FBS or FBS pre-screened for MSC-culture, hereon FBS-MSC). MSC identity was confirmed by flow cytometry (CD90+, CD44+, MHC-II−) and multilineage differentiation assays. Upon isolation, cells showed typical MSC morphology and ≥ 95

Liver-on-a-chip models predictive for both metabolism, and blood and canalicular transport of drug candidates in humans are lacking. Here, we established a bioengineered and 3Rs-complied (animal component-free) hepatocyte-like millifluidic system based on 3D hollow fiber membranes (HFMs), recombinant human laminin 332 coating and adult human stem cell-derived organoids. Organoid fragments formed polarized and tight monolayers on HFMs with improved hepatocyte-like maturation, as compared to standard 3D organoid cultures in Matrigel from matched donors. Gene expression profiling and immunofluorescence revealed that hepatocyte-like monolayers expressed a broad panel of phase I (e.g. CYP3A4, CYP2D6, CYP2C9) and II (e.g. UGTs, SULTs) drug-metabolizing enzymes and drug transporters (e.g. MDR1, MRP3, OATP1B3). Moreover, statically cultured monolayers displayed phase I and II metabolism of a cocktail of six relevant compounds, including midazolam and 7-hydroxycoumarin. We also demonstrated the disposition of midazolam in the basal/blood-like circulation and apical/canalicular-like compartment of the millifluidic chip. Finally, we studied the bioavailability of midazolam and coumarin on-a-chip in combination with a small intestine-like system. In conclusion, we generated a proof-of-concept liver organoid-on-a-chip model for examining metabolism and transport of drugs, which can be further developed to predict pharmacokinetics' (PK)/absorption, distribution, metabolism and excretion (ADME) profiles in humans.

Copper-associated hepatitis is a hereditary disease in Labrador Retrievers with a complex genetic background. Currently, liver biopsies are needed for diagnosis and treatment monitoring. A serum-based biomarker for hepatic copper levels could provide a less invasive diagnostic approach. Circulating microRNAs are increasingly studied for their diagnostic potential in hepatobiliary disease and could be utilized in assessing hepatic copper levels. Currently, information on the potential use of copper specific microRNAs in dogs is lacking. The aim of this pilot study was to identify microRNAs associated with elevated hepatic copper levels in Labrador Retrievers. Client-owned Labrador Retrievers with normal (n = 15) and elevated hepatic copper levels (n = 21) were retrospectively selected from a patient database. We performed a microRNA screening array of 277 microRNAs in blood serum of Labrador Retrievers with normal (n = 5) and elevated hepatic copper levels (n = 5). MicroRNAs upregulated in Labrador Retrievers with elevated hepatic copper were subsequently analyzed with qPCR in a replication cohort of Labrador Retrievers with normal (n = 13) and elevated (n = 18) hepatic copper levels. Results showed that six out of the 277 serum microRNAs were significantly upregulated in dogs with elevated hepatic copper and these were analyzed in the replication cohort. After Bonferroni correction, cfa-miR-30b (fold-change 2.17, p-value .002) was significantly upregulated in the replication cohort. In this exploratory study, cfa-miR-30b is increased in Labrador Retrievers with elevated hepatic copper levels. This result justifies validation in a larger cohort of dogs with and without hepatic copper accumulation, including those with different forms of liver disease and other breeds affected by copper-associated hepatitis.

Cholangiocyte organoids provide a powerful platform for applications ranging from in vitro modeling to tissue engineering for regenerative medicine. However, their expansion and differentiation are typically conducted in animal-derived hydrogels, which impede the full maturation of organoids into functional cholangiocytes. In addition, these hydrogels are poorly defined and complex, limiting the clinical applicability of organoids. In this study, a novel medium composition combined with synthetic polyisocyanopeptide (PIC) hydrogels to enhance the maturation of intrahepatic cholangiocyte organoids (ICOs) into functional cholangiocytes is utilized. ICOs cultured in the presence of sodium butyrate and valproic acid, a histone deacetylase inhibitor, and a Notch signaling activator, respectively, in PIC hydrogel exhibit a more mature phenotype, as evidenced by increased expression of key cholangiocyte markers, crucial for biliary function. Notably, mature cholangiocyte organoids in PIC hydrogel display apical-out polarity, in contrast to the traditional basal-out polarization of ICOs cultured in Matrigel. Moreover, these mature cholangiocyte organoids effectively model the biliary pro-fibrotic response induced by transforming growth factor beta. Taken together, an animal-free, chemically defined culture system that promotes the ICOs into mature cholangiocytes with apical-out polarity, facilitating regenerative medicine applications and in vitro studies that require access to the apical membrane, is developed.

For over 150 years, researchers have studied the (patho)physiology of the endocrine pancreas and devised treatment options for diabetes mellitus (DM). However, no cure has been developed so far. In dogs, diabetes mellitus type 1 (T1DM) is the most common presentation. Treatment consists of twice daily insulin injections, monitored by spatial blood glucose measurements. Even though dogs were instrumental in the discovery of insulin and islet transplantations, the treatment in diabetic dogs has remained unchanged for decades. Providing twice daily insulin injections is demanding for both owners and dogs and may result in hypoglycaemic events, creating the need for new treatment strategies. Novel regenerative medicine-based tools, such as improved β-cell culture protocols and artificial devices, have sparked hope for a cure. In human medicine, emerging technologies such as the transplantation of insulin-producing β-cells, generated by stem cell differentiation, with or without an encapsulation device, are currently tested in phase I/II clinical trials. As the pathogenesis of T1DM is remarkably similar between humans and dogs, novel treatment methods could be implemented in canine medicine. This review briefly summarises the physiology of the canine endocrine pancreas and the pathophysiology of canine DM before exploring current and possible future treatment options for canine DM.

The application of liver organoids is very promising in the field of liver tissue engineering; however, it is still facing some limitations. One of the current major limitations is the matrix in which they are cultured. The mainly undefined and murine-originated tumor matrices derived from Engelbreth-Holm-Swarm (EHS) sarcoma, such as Matrigel, are still the standard culturing matrices for expansion and differentiation of organoids toward hepatocyte-like cells, which will obstruct its future clinical application potential. In this study, we exploited the use of newly developed highly defined hydrogels as potential matrices for the culture of liver organoids and compared them to Matrigel and two hydrogels that were already researched in the field of organoid research [i.e., polyisocyanopeptides, enriched with laminin-entactin complex (PIC-LEC) and gelatin methacryloyl (GelMA)]. The newly developed hydrogels are materials that have a physicochemical resemblance with native liver tissue. Norbornene-modified dextran cross-linked with thiolated gelatin (DexNB-GelSH) has a swelling ratio and macro- and microscale properties that highly mimic liver tissue. Norbornene-modified chondroitin sulfate cross-linked with thiolated gelatin (CSNB-GelSH) contains chondroitin sulfate, which is a glycosaminoglycan (GAG) that is present in the liver ECM. Furthermore, CSNB-GelSH hydrogels with different mechanical properties were evaluated. Bipotent intrahepatic cholangiocyte organoids (ICOs) were applied in this work and encapsulated in these materials. This research revealed that the newly developed materials outperformed Matrigel, PIC-LEC, and GelMA in the differentiation of ICOs toward hepatocyte-like cells. Furthermore, some trends indicate that an interplay of both the chemical composition and the mechanical properties has an influence on the relative expression of certain hepatocyte markers. Both DexNB-GelSH and CSNB-GelSH showed promising results for the expansion and differentiation of intrahepatic cholangiocyte organoids. The stiffest CSNB-GelSH hydrogel even significantly outperformed Matrigel based on ALB, BSEP, and CYP3A4 gene expression, being three important hepatocyte markers.

Accurate liver disease modeling and drug toxicity testing still remain challenging as liver cells in vitro poorly resemble adult hepatocytes, as we previously demonstrated using whole transcriptome and cell identity analysis. To address this, we used our insights into hepatic modeling to develop hepatocyte-like liver organoids (HeLLOs), a novel human organoid model with mature hepatocyte functions superior to existing models. HeLLOs are easily established from (small) healthy or diseased liver tissues and rapidly expanded for an extended period in optimized culture conditions. Transcriptomic and functional analyses revealed that differentiated HeLLOs closely resemble fresh primary human hepatocytes (PHHs) and perform key hepatic functions such as gluconeogenesis, drug metabolism, and bile acid synthesis. We developed a HeLLO-based toxicity assay with higher sensitivity in predicting liver toxicity of known liver-toxic drugs compared to the gold-standard PHHs. By modeling disease-related mechanisms, such as bile acid transport, HeLLOs uncover transport-inhibition toxicity mechanisms of known liver toxic drugs. Single cell sequencing analysis of HeLLOs identified a heterogeneous cluster of cells with cholangiocyte-like and hepatocyte-like cells, overall resembling liver regenerative cells. As such, HeLLOs hold great promise for advancing liver disease modeling and drug testing. To our knowledge, HeLLOs are the best expandable liver model for predicting adverse drug reactions as well as modeling various liver disease mechanisms. ### Competing Interest Statement A.I.A, I.P.J, I.F.S, and S.A.F. are inventors on the patent describing the hepatocyte-like liver organoids (HeLLOs).

Drug induced bile duct injury is a frequently observed clinical problem leading to a wide range of pathological features. During the past decades, several agents have been identified with various postulated mechanisms of bile duct damage, however, mostly still poorly understood. Here, we investigated the mechanisms of chlorpromazine (CPZ) induced bile duct injury using advanced in vitro cholangiocyte cultures. Intrahepatic cholangiocyte organoids (ICOs) were driven into mature cholangiocyte like cells (CLCs), which were exposed to CPZ under cholestatic or non-cholestatic conditions through the addition of a bile acid cocktail. CPZ caused loss of monolayer integrity by reducing expression levels of tight junction protein 1 (TJP1), E-cadherin 1 (CDH1) and lysyl oxidase homolog 2 (LOXL2). Loss of zonula occuludens-1 (ZO-1) and E-cadherin was confirmed by immunostaining after exposure to CPZ and rhodamine-123 leakage further confirmed disruption of the cholangiocyte barrier function. Furthermore, oxidative stress seemed to play a major role in the early damage response by CPZ. The drug also decreased expression of three main basolateral bile acid transporters, ABCC3 (ATP binding cassette subfamily C member 3), SLC51A/B (solute carrier family 51 subunit alpha/beta) and multidrug resistance transporter ABCB1 (ATP binding cassette subfamily B member 1), thereby contributing to bile acid accumulation. CPZ did not induce an inflammatory response by itself, but addition of TNFα revealed a synergistic effect. These results show that ICOs present a model to identify toxic drugs affecting the bile ducts while providing mechanistic insights into hepatotoxicity.

Background: To accurately measure permeability of compounds in the intestine, there is a need for preclinical in vitro models that accurately represent the specificity, integrity and complexity of the human small intestinal barrier. Intestine-on-chip systems hold considerable promise as testing platforms, but several characteristics still require optimization and further development. Methods: An established intestine-on-chip model for tissue explants was adopted for intestinal cell monolayer culture. A 3D-printed culture disc was designed to allow cell culture in static conditions and subsequent permeability studies in a dynamic environment. Membrane characteristics and standardized read-outs were investigated and compared to traditional permeability studies under static conditions. Results: By starting cultures outside the chip in conventional wells plates, the new cell disc design could support accurate cell monolayer formation for both Caco-2 and human enteroids. When transferred to the chip with laminar flow, there was accurate detection of barrier integrity (FD4 and Cascade Blue) and permeability (atenolol/antipyrine). Both flow and membrane characteristics had a significant impact on permeability outcomes. Conclusions: This novel intestinal cell-on-chip system offers large flexibility for intestinal permeability studies, although it still requires validation with more compounds to reveal its full potential.

Conventional static culture of organoids necessitates weekly manual passaging and results in nonhomogeneous exposure of organoids to nutrients, oxygen, and toxic metabolites. Here, we developed a miniaturized spinning bioreactor, RPMotion, specifically optimized for accelerated and cost-effective culture of epithelial organoids under homogeneous conditions. We established tissue-specific RPMotion settings and standard operating protocols for the expansion of human epithelial organoids derived from the liver, intestine, and pancreas. All organoid types proliferated faster in the bioreactor (5.2-fold, 3-fold, and 4-fold, respectively) compared to static culture while keeping their organ-specific phenotypes. We confirmed that the bioreactor is suitable for organoid establishment directly from biopsies and for long-term expansion of liver organoids. Furthermore, we showed that after accelerated expansion, liver organoids can be differentiated into hepatocyte-like cells in the RPMotion bioreactor. In conclusion, this miniaturized bioreactor enables work-, time-, and cost-efficient organoid culture, holding great promise for organoid-based fundamental and translational research and development.

Adult primary human hepatocytes (PHHs) are the gold standard in ex vivo toxicological studies and possess the clinical potential to treat patients with liver disease as advanced therapy medicinal products (ATMPs). However, the utility of this valuable cell type has been limited by short-term functionality and limited expansion potential in vitro . While notable advances have been made in the long-term maintenance of primary hepatocytes, there has been limited success in driving the efficient generation and expansion of adult PHH-derived organoids which recapitulate both liver tissue architecture and function, hampering in vitro studies and regenerative medicine applications. Here we describe the mass generation and long-term expansion of hepatobiliary organoids with functionally interconnected hepatic and biliary-like structures from adult primary human hepatocytes. Hepatobiliary organoids retain the expression of lineage and functional markers, closely resembling PHH, while also acquiring the expression of regeneration, fetal and biliary markers. Organoids perform key hepatocyte functions while proliferating and can be matured to enhance their functionality. As a proof-of-principle, we demonstrate that hepatobiliary organoids can recapitulate hallmarks of cholestasis and steatosis in vitro . Moreover, we show that hepatocytes can be transfected, transduced and gene edited in 3D prior to organoid generation, facilitating a wide range of applications. Our novel hepatobiliary organoid system bridges the gap between short-term functionality of primary human hepatocytes and the need for scalable, long-term organoid models of the adult liver, offering immense potential for drug testing, disease modeling, and advanced therapeutic applications. ### Competing Interest Statement The authors have declared no competing interest.

End-stage liver diseases have an increasing impact worldwide, exacerbated by the shortage of transplantable organs. Recognized as one of the promising solutions, tissue engineering aims at recreating functional tissues and organs in vitro. The integration of bioprinting technologies with biological 3D models, such as multi-cellular spheroids, has enabled the fabrication of tissue constructs that better mimic complex structures and in vivo functionality of organs. However, the lack of methods for large-scale production of homogeneous spheroids has hindered the upscaling of tissue fabrication. In this work, we introduce a fully automated platform, designed for high-throughput sorting of 3D spheroids based on label-free analysis of brightfield images. The compact platform is compatible with standard biosafety cabinets and includes a custom-made microscope and two fluidic systems that optimize single spheroid handling to enhance sorting speed. We use machine learning to classify spheroids based on their bioprinting compatibility. This approach enables complex morphological analysis, including assessing spheroid viability, without relying on invasive fluorescent labels. Furthermore, we demonstrate the efficacy of transfer learning for biological applications, for which acquiring large datasets remains challenging. Utilizing this platform, we efficiently sort mono-cellular and multi-cellular liver spheroids, the latter being used in bioprinting applications, and confirm that the sorting process preserves viability and functionality of the spheroids. By ensuring spheroid homogeneity, our sorting platform paves the way for standardized and scalable tissue fabrication, advancing regenerative medicine applications.