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Cited by 3293D Printing in Biomedical ResearchTissue Engineering and Regenerative MedicinePluripotent Stem Cells Research

In situ expansion, differentiation, and electromechanical coupling of human cardiac muscle in a 3D bioprinted, chambered organoid

Open access
Kupfer, Molly E., Lin, Wei-Han, Ravikumar, Vasanth, Qiu, Kaiyan, Wang, Lu, Gao, Ling, Bhuiyan, Didarul B., Lenz, Megan +7 more·2020·Circulation Research
DOI 10.1161/circresaha.119.316155PMC PMC8210857
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Abstract

RATIONALE: One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes-a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. OBJECTIVE: To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. METHODS AND RESULTS: We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell-laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. CONCLUSIONS: This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.

Integrity signals

Registry

Crossref · 10.1161/circresaha.119.316155

Cites

63 of 63 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

59 of 78 cited sources checked · 19 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Stem cell maturation, differentiation, and derived cell function depend critically on temporal and spatial engagement of cell surface integrins with ECM in development and ex vivo culture.

    “There is now clear evidence from our group and the work of others that stem cell maturation and differentiation and the proper function of cells derived thereof are critically dependent on the temporal and spatial engagement of cell surface integrins with extracellular matrices (ECM) both in a given organ system during development19 and in the context of ex vivo stem cell culture20–27.”
    Quote in the paperSubstantial claim4 of 9 sources checked
    • cites [19] source
    • cites [20] source

      The Introduction (page not provided) says integrin-mediated interactions are vital to normal cell function and differentiation, and the Results & Discussion report changes in integrin expression as human embryonic stem cells form definitive endoderm. The paper does not establish that maturation and the function of derived cells critically depend on the temporal and spatial engagement of integrins with ECM; it describes the proposed role of the observed integrins as requiring further investigation. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [21] source

      The Abstract and Discussion (page numbers not supplied) show that integrin engagement with fibronectin and vitronectin affects human embryonic stem-cell differentiation to definitive endoderm in culture. This supports the ex vivo differentiation part of the citation, but the paper does not establish dependence of later maturation or derived-cell function on temporal and spatial integrin–ECM engagement. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [22] source
    • cites [23] source
    • cites [24] source
    • cites [25] source

      The Introduction (page unavailable) says extracellular substrates, including ECM, can guide ex vivo stem-cell growth and differentiation. The Results and Discussion (page unavailable) show that integrins largely mediate human ES-cell adhesion to ECM-coated surfaces. The paper does not establish that temporal and spatial integrin–ECM engagement is critical for maturation, differentiation, or derived-cell function; it also reports undifferentiated growth on surfaces that do not act through integrins. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [26] source
    • cites [27] source

      The paper’s Discussion (page not provided) reports that α5β1 integrin priming promotes osteogenic differentiation of cultured mesenchymal cells, supporting the claim’s ex vivo differentiation component. It does not establish that stem cell maturation or derived-cell function generally depends on temporal and spatial integrin–ECM engagement; its model promotes differentiation independently of the cell adhesion mediated by the peptide’s RRET sequence. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Premisecitation

    3D bioprinting can generate models replicating the heart's complex structure with chambers and large vessels using soft biomaterials.

    “Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3D bioprinting.”
    • Also stated in: § INTRODUCTION · hedged
      “3D bioprinting has been proposed as a means to generate more geometrically complex tissues from the bottom-up.”
    Quote in the paperSubstantial claim7 of 9 sources checked
    • cites [9] source

      The Results and Discussion (Fig. 4; page unavailable) describe printing a complex embryonic chick-heart model in soft alginate, and the Methods identify atria and ventricles in the heart used to make the model. The paper describes a coronary arterial tree as a separate print (Fig. 3), not large vessels reproduced within the printed heart model. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [10] source

      The Introduction (page not provided) says bioprinting produces controlled 3D structures with defined biomaterial geometries. The Results and Discussion (page not provided) describe cylindrical cardiac patches, not models replicating a whole heart with chambers and large vessels. That specific anatomical claim is unsupported. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [11] source

      The 3D Printing section (page not provided) describes patient-specific heart models made from a printed scaffold infused with silicone and hydrogels to mimic myocardial tissue. The Segmentation and Digital Modeling section identifies structures including the aorta and ventricular septum, but the paper does not establish that the models replicate the heart’s full chamber-and-large-vessel structure. It describes 3D printing, not bioprinting. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [12] source

      The Abstract and 'FRE Printing 3D PDMS Structures' section describe printing complex structures from soft PDMS, including tubes and a bifurcation (page not specified in our copy). They do not report a heart model or a structure replicating the heart’s chambers and large vessels. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [13] source

      The Methods (page unavailable) describe 3D-printed aortic models made with flexible resin, supporting the narrower point that flexible cardiac-anatomy models can be printed. They do not describe 3D bioprinting with soft biomaterials or a model of the heart with chambers and large vessels; the left atrium and ventricle were explicitly excluded. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [14] source

      The Development of Printing Materials section (page not provided) says PolyJet 3D printing can replicate complex cardiac anatomy using materials ranging from very soft to hard. The Future Directions section (page not provided) also discusses modeling vessel and chamber walls with material blends. The copy does not establish that 3D bioprinting, specifically, produces a model of the heart incorporating both chambers and large vessels from soft biomaterials. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [15] source
    • cites [16] source

      The Current applications section describes 3D-printed models showing complex relationships among cardiac chambers and great arteries, and the 3D printing process section says some printers make soft, rubberlike models (page numbers unavailable). But the paper distinguishes those anatomical models from bioprinting, which it discusses in the Abstract and Future direction as a prospect for implants and tissue engineering. It does not say the described heart models are made by 3D bioprinting. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [17] source
  • Premisereasoning

    Macroscale contractile function has not yet been achieved in a 3D printed, perfusable, chambered heart model.

    “The fact that macroscale contractile function has not yet been achieved in a 3D printed, perfusable, chambered heart model reflects the challenges associated with handling mature cardiac muscle cells.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [8] source

      The passage indexed as Abstract (page unavailable) says a fully functional 3D-bioprinted organ has not yet been achieved, and describes perfusable vasculature separately from a contracting ventricular model. But that model did show chamber-scale contraction, including a roughly 5% decrease in interior cross-sectional area. The paper therefore supports the absence of a demonstrated combination of perfusion and contraction in its own chambered model, not an unqualified claim that macroscale contraction has not been achieved. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [9] source

      In Results and Discussion (page not available), the paper describes a macroscale heart-shaped alginate print with anatomical detail, while perfusion is demonstrated in a separate printed coronary arterial tree. This supports that the paper did not demonstrate a perfusable, contracting chambered heart model, but it does not establish the broader claim that no such model had yet been achieved. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [18] source
  • Premisereasoning

    Inclusion of contiguous living muscle supporting pump function in bioprinted heart models has not been achieved.

    “Yet, inclusion of contiguous, living muscle to support pump function has not been achieved.”
    • Also stated in: § INTRODUCTION · plain
      “The fact that macroscale contractile function has not yet been achieved in a 3D printed, perfusable, chambered heart model reflects the challenges associated with handling mature cardiac muscle cells.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [8] source

      The ventricle-model passage (section labeled Abstract; page unavailable) reports a viable, dense layer of interconnected cardiomyocytes that contracted and reduced the chamber’s cross-sectional area. The concluding passage says a fully functional bioprinted organ has not yet been achieved, but the paper does not establish that contiguous living muscle has not been included in a bioprinted heart model. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [9] source

      In Results and Discussion, the printed heart is described as a fluorescently labeled alginate model made from an image of a fixed embryonic chick heart, not a living, pumping construct. This supports the limitation for this paper’s heart model, but the paper does not establish the broader claim that no bioprinted heart model has achieved contiguous living muscle supporting pump function. Page numbers are not available in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [18] source
  • Premisecitation

    The only 3D printed volume-holding structure that has demonstrated cardiac function is a single ventricle model.

    “In fact, the only 3D printed, volume-holding structure to successfully demonstrate cardiac function is a single ventricle model.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [8] source

      The paper’s ventricle-model passage (labeled Abstract in our copy; no page given) describes a printed chamber that maintained its shape and contracted. A later passage reports a decrease in its interior cross-sectional area during contraction. These passages support the functional single-ventricle example, but do not establish that it was the only 3D-printed volume-holding structure to demonstrate cardiac function. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • May combine two claims. 41
  • Classified as probably not a substantial claim. 10
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    The hChaMP maintained its functions for at least six weeks, whereas most comparable models have been characterized only up to two weeks after fabrication.

    “Notably, we show that the hChaMP can maintain these functions for at least 6 weeks, while most similar models have been characterized only up to 2 weeks after fabrication6–8, 18.”
    Quote in the paperSubstantial claim4 of 4 sources checked
    • cites [6] source

      The Materials and methods describe functional testing of this paper’s ventricular chamber after 10–12 days in culture, which supports it as an example of a model characterized around two weeks. The Discussion also describes improved function with culture extended to day 20. Neither passage establishes that most comparable models were characterized only up to two weeks; the six-week result is the citing authors’ own finding, not a claim attributed to this source. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [7] source
      Experimental — accuracy not yet measured
    • cites [8] source

      The source’s ventricle-model passage (indexed as Abstract; no page supplied) says its printed ventricles were cultured for up to 28 days, not only two weeks. It does not establish how long most other models were characterized, and the citing authors’ six-week result is their own finding rather than one attributed to this source. (LLM, conf=0.78)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [18] source
      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Researchers have generated volume-handling cardiac tissue models that recapitulate pressure-volume dynamics of the heart.

    “To address this issue, researchers have made strides in the generation of volume-handling cardiac tissue models capable of recapitulating pressure-volume dynamics of the heart6–8.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [6] source
    • cites [7] source
    • cites [8] source

      The ventricle-model passage (labeled Abstract in our copy; no page given) reports a contracting printed chamber whose cross-sectional area decreased by up to about 5% at peak systole. This supports a cardiac model with changing chamber dimensions, but the paper does not show that it recapitulated the heart’s pressure-volume dynamics. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    These volume-handling models are limited to a single ventricle and lack perfusion capacity.

    “However, these tissues are limited to a single ventricle model and thus lack the capacity for perfusion.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [6] source

      The Introduction and Discussion describe a simplified, single ventricle-like chamber, supporting the single-ventricle part of the claim. The Discussion contrasts the model with native heart tissue that has integrated vasculature, but the paper does not establish that being a single-ventricle model causes a lack of perfusion capacity. The Results also report that the chamber pumps fluid, so lack of perfusion should not be read as lack of fluid movement. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [7] source

      The Model assumptions section describes a single left-ventricle model with an unvascularized wall, supporting the lack of vascular tissue perfusion. But the Bioreactor section reports fluid flow through the ventricle and demonstrates assisted, valve-directed pumping. The paper therefore does not support the broader claim that a single-ventricle model lacks perfusion capacity altogether. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [8] source

      The ventricle-model passage (labeled Abstract in our copy; page unavailable) describes a printed left ventricle, supporting the single-ventricle part. It does not establish that this model lacks the capacity for perfusion. A separate passage under the same label reports successful perfusion of a printed vascular network, though not of the ventricle model. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Early engineered heart tissues were simple strips or rings made by casting cardiomyocytes in ECM-based gel.

    “Early engineered heart tissues consisted of geometrically simple structures (strips or rings) made by casting cardiomyocytes in an extracellular matrix-based gel1–3.”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [1] source
    • cites [2] source
    • cites [3] source

  • May combine two claims.

    Premisecitation

    Multiple studies have printed entire heart organ models with biological materials, but the constructs either lacked cells or evidence of electromechanical function.

    “Multiple studies have demonstrated the capacity to print entire heart organ models using biological materials, but the resulting constructs have either lacked cells8, 9 or evidence of electromechanical function18.”
    Quote in the paperSubstantial claim3 of 3 sources checked

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Printing tissues composed entirely of native proteins, cells, and/or biocompatible synthetic components is possible and accessible to many laboratories.

    “The concept is gaining traction, as the ability to print tissues composed entirely of native proteins, cells and/or biocompatible synthetic components is possible and accessible to many laboratories9–17.”
    Quote in the paperNot a substantial claim7 of 9 sources checked
    • cites [9] source

      The Results and Discussion (page unavailable) demonstrate printing biological structures from natural biopolymers and describe a low-cost FRESH setup intended to make bioprinting accessible to more laboratories. But the authors say that directly bioprinting functional tissues and organs still requires further development; they anticipate broader laboratory access rather than establish that it has already been achieved. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [10] source

      The Conclusions (page not provided) report successful bioprinting of a cardiac patch made with extracellular matrix, progenitor cells, and gelatin methacrylate. This establishes that such printing is possible, but the paper does not establish that it is accessible to many laboratories. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [11] source

      The 3D Printing section (page not provided) describes making a heart model from a proprietary scaffold, silicone, and hydrogels, supporting the narrower point that tissue-like models can be printed. It does not establish that the model is tissue composed of native proteins or cells, that its synthetic components are biocompatible, or that this process is accessible to many laboratories. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [12] source

      The Introduction describes PDMS as a biocompatible biomaterial, and the paper demonstrates printing PDMS structures. The Abstract says its low-cost, open-source approach can be rapidly implemented for research applications. It does not demonstrate printing tissues composed entirely of native proteins, cells, or synthetic components, or establish that tissue printing is accessible to many laboratories. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [13] source

      The Methods (Creation of 3D Models and Valves; page unavailable) describe printing anatomical models in flexible resin and hard plastic, not printing tissues. A search of our copy for native proteins, cells, biocompatible components, and laboratory accessibility found no passage addressing whether such tissue printing is possible or accessible to many laboratories. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
    • cites [14] source

      The Printing Technology and Development of Printing Materials sections describe synthetic materials used to print anatomical models that approximate cardiac tissue properties. They do not address printing tissues made of native proteins or cells, whether the synthetic components are biocompatible, or whether such printing is accessible to many laboratories (searched: native proteins, cells, bioprinting, laboratories). (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [15] source
    • cites [16] source

      The Future direction section (page unavailable) says 3D printing has been used experimentally for tissue engineering, with cells placed on a printed scaffold. It does not establish that tissues composed entirely of the listed components can be printed or that this is accessible to many laboratories; the Current limitations section (page unavailable) instead describes expensive equipment and potentially prohibitive costs for small programs. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [17] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Fibronectin/integrin β1/β-catenin signaling promotes mesoderm emergence from induced pluripotent stem cells.

    “Finally, fibronectin/integrin β1/β−catenin signaling promotes the emergence of mesoderm from induced pluripotent stem cells.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] source

      The Discussion (page unavailable) says fibronectin signals through integrin-β1 and stimulates Wnt/β-catenin signaling to promote mesodermal fate. However, the Materials and Methods identify the cells studied as mouse embryonic stem cells, not induced pluripotent stem cells; the claimed cell type is unsupported. (LLM, conf=0.91)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A study first established a direct link between integrin-linked kinase (ILK) and GSK3β, the primary β-catenin antagonist.

    “This study was the first to establish a direct link between elements of the focal adhesion, namely integrin-linked kinase (ILK), with GSK3β29, the primary antagonist of β-catenin.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] source

      The Results (page unavailable, Fig. 4D) report that Gsk3β co-immunoprecipitated with ILK, supporting a link between them. The paper does not establish that it was the first study to find that link, and co-immunoprecipitation alone does not establish direct binding. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationown data

    The 10G0.25CFL gels were elastic with stiffness similar to the late embryonic heart.

    “suggesting the elastic nature of the gels with a stiffness similar to that of the late embryonic heart31.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [31] source

      The Results and Discussion section reports that embryonic hearts behave elastically and stiffen to neonate and adult heart stiffness by embryonic day 14 (page unavailable). It does not describe 10G0.25CFL gels or measure their elasticity or stiffness, so it cannot establish the claimed similarity. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Type III collagen engages at least integrins α1β1 and α2β1, which are highly expressed in iPSC-derived cardiomyocytes.

    “which will engage at least integrins α1β1 and α2β1, which are highly expressed in iPSC-derived cardiomyocytes38.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [38] source

      The Introduction and Results describe gene-expression profiling of hiPSC-derived cardiomyocytes, but the passages retrieved for integrins α1β1 and α2β1, ITGA1/ITGA2/ITGB1, and type III collagen do not address those integrins’ expression or collagen binding. Our copy therefore does not verify the claim attributed to this paper. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    iPSC-derived cardiomyocytes express many MMPs, including 1-3, 10, 11, 14-16, and 19, and hiPSC-derived smooth muscle cells, which were present in the hChaMP though less common, do so as well.

    “iPSC-derived cardiomyocytes express a myriad of MMPs including 1–3, 10, 11, 14–16 and 1938, as do hiPSC-derived smooth muscle cells, which were present, albeit more rarely in the hChaMP.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [38] source

      The Results section on emerging cellular heterogeneity reports gene-expression profiles in hiPSC-derived cardiomyocytes, but does not identify the claimed MMPs. Searches for MMP19, the listed MMP genes, and matrix metalloproteinases found no supporting passage in our copy. The claim about smooth muscle cells and hChaMP is likewise not addressed. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation

    The hChaMP generated a low pressure of 0.23 mmHg, which is higher than other reported values.

    “the pressure generated by the hChaMP, while low (0.23 mmHg), is higher than other reported values6, 7.”
    Quote in the paperSubstantial claim2 of 2 sources checked

  • May combine two claims.

    Premisecitation

    Among the relevant integrins, only α6β1 has been reported to support pluripotency, whereas the others appear to promote differentiation in an ECM-specific way.

    “only α6β1 has been reported to promote pluripotency37, while all others appear to promote differentiation in an ECM type-specific manner.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [37] source

      The Discussion (page unavailable) supports a role for α6β1 in maintaining human pluripotent stem-cell self-renewal. It does not support the claim that all other integrins promote ECM-specific differentiation: the Introduction says their roles remain incompletely understood, and the paper’s reference list identifies a study of self-renewal via αVβ5. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationown data

    The thinnest hChaMP walls match prior literature values, while the thickest walls greatly exceed existing tissue models.

    “Wall thicknesses on the low end of this spectrum match what has been previously demonstrated in the literature, and the high end of the spectrum far exceeds existing tissue models6–8.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [6] source

      The Results section (Bioengineering hvCOC; page unavailable) reports a wall thickness of approximately 100 μm. It does not compare that thickness with prior wall-thickness values or establish that thicker hChaMP walls exceed existing tissue models. The cited paper also studies hvCOCs, not the citing document’s hChaMPs. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [7] source

      In “Model assumptions for tissue-engineered ventricle chambers” (page unavailable), MacQueen et al. describe a tissue-engineered ventricle designed with a wall thickness limited to approximately 0.1 mm, providing a prior thin-wall benchmark. The paper does not establish that the thickest hChaMP walls exceed existing tissue models, so it supports only the low-end comparison. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [8] source

      The passage on the FRESH-printed coronary artery tube (section labeled Abstract; no page given) reports a wall thickness of approximately 300 μm, providing a prior-literature benchmark for thin printed walls. It does not give the hChaMP thickness range or establish that its thickest walls greatly exceed existing tissue models. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    The resulting hChaMPs showed macroscale beating and continuous action potential propagation and responded to drugs and pacing.

    “hChaMPs demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The connected chambers permitted perfusion and allowed replication of pressure/volume relationships relevant to heart function and remodeling in health and disease.

    “The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In some hChaMPs, electrical point stimulation at another site overrode the spontaneous depolarization source and changed the direction of propagation.

    “However, in some cases the spontaneous source of depolarization could be overcome and the directionality of propagation altered via electrical point stimulation at another location within the hChaMP”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    All tested ink formulations had viscosities within 10-40 cP, ensuring low pressure requirements (28-38 kPa) during printing.

    “We found, according to design, that all of the ink formulations fell within the range of 10–40 cP, ensuring low pressure requirements (28–38 kPa) with printing.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Differentiation outcomes corresponded to GelMA fraction and were largely dependent on fibronectin and laminin addition.

    “We found that differentiation outcomes not only corresponded to the GelMA fraction, but also were largely dependent on the addition of fibronectin and laminin (Table 1, Figure 2d, e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The hChaMPs were routinely produced and showed macroscale beating.

    “hChaMPs could be routinely fabricated such that macroscale beating was observed (Online Video V).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For crosslinked 10G0.25CFL gels, storage modulus was 6.14 ± 1.13 kPa and loss modulus was 0.09 ± 0.06 kPa.

    “For the crosslinked gels of this specific formulation, the storage modulus (G’, 6.14 ± 1.13 kPa) was nearly two orders of magnitude higher than the loss modulus (G”, 0.09 ± 0.06 kPa)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Particles of approximately 100 μm supported printing of the chambered cardiac mimic with intact chambers, patent large vessels, and handleable material properties.

    “Particles of approximately 100 μm could support printing of the chambered cardiac mimic with intact chambers, patent large vessels, and material properties conducive to handling (Online Video III).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GelMA below 10% produced flimsy, easily damaged structures, while above 15% required high pressures that made material control difficult and sometimes damaged cells.

    “Material handling was difficult below 10% GelMA as final structures were flimsy and easily damaged; whereas printability was difficult to accomplish above 15% GelMA as extrusion required high pressures which made material control difficult and which sometimes resulted in cell damage.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Intracellular machinery associated with the sarcolemma and sarcoplasmic reticulum was well expressed and appropriately organized.

    “Intracellular machinery associated with the sarcolemma and sarcoplasmic reticulum was also well expressed and appropriately organized (Figure 5c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Contractile performance did not improve over time and did not decline significantly.

    “Contractile performance was not enhanced over time but did not decline significantly either”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The hChaMP wall thickness was typically 100-500 μm, with the thickest region exceeding 500 μm.

    “and the thickness of the wall was typically between 100 μm and 500 μm with the thickest region exceeding 500 μm (Figure 4e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Bioink was discernible in the inner portion of the hChaMP wall but was largely without cells.

    “Bioink could be discerned in the inner portion of the hChaMP wall, but was largely without cells (Figure 4e, white oval).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The 10G0.25CFL formulation achieved cell density of 0.1 mg DNA/g gel, on the same order as native cardiac tissue (~0.3 mg DNA/g myocardial mass) and significantly higher than the lowest-differentiation condition.

    “In addition, this formulation achieved a cell density of 0.1 mg DNA/g of gel, which is on the same order of magnitude of native cardiac tissue, (~0.3 mg DNA/g of myocardial mass) and significantly higher than the condition that produced the lowest amount of differentiation32”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [32] source

  • May combine two claims.

    Findingown data

    A sacrificial ink backfilling approach was developed but low cell viability and proliferation associated with the sacrificial ink limited further use.

    “To address this hurdle, we developed an approach that involved backfilling an inverted geometry made of sacrificial ink; however, low cell viability and low proliferation associated with the sacrificial ink limited further use”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    SERCA2a signal was present in most cells and closely apposed to myofibrils (1.50 ± 0.19% of cTnT area).

    “Sarcoplasmic reticulum ATPase2a (SERCA2a) exhibited signal in the majority of cells, with staining in close apposition to the myofibrils (1.50 ± 0.19% of cTnT area).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    By Day 14, about 90% of the bioink volume was populated with cells, singular and in large colonies, with 40% ± 11% colony area.

    “By Day 14, approximately 90% of the bioink volume was populated with cells, both singular and in large colonies, with 40% ± 11% corresponding to colony area (Figure 4a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Smooth muscle cells (10.5 ± 4.2% via αSMA) and endothelial cells (4.2 ± 2.4% via CD31) were present in hChaMPs.

    “but smooth muscle cells (10.5 ± 4.2% of cells via staining for alpha smooth muscle actin, αSMA) and endothelial cells (4.2 ± 2.4% of cells via staining for CD31) were present in hChaMPs.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    The end result is a living pump mimicking native heart chambers and large vessel conduits while housing viable, densely packed, functional cardiomyocytes.

    “The end result is a living pump that mimics the chambers and large vessel conduits of a native heart while housing viable, densely packed and functional cardiomyocytes (Figure 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The typical volume moved through the hChaMP chambers was 0.5 μL, and the maximum was 5.0 μL.

    “The usual volume moved through the chambers was 0.5 μL and maximum volume moved through the chambers was 5.0 μL”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    The expansion phase produced final cell densities of about 0.1 mg DNA per g of hChaMP, within the same order of magnitude as native myocardial tissue at about 0.3 mg DNA per g.

    “This critical expansion phase enabled final cell densities (~0.1 mg DNA/g of hChaMP) of the same order of magnitude as native tissue (~0.3 mg DNA/g of myocardial mass)32.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [32] source

  • May combine two claims.

    Findingreasoning

    The calculated ejection fraction averaged 0.7% and reached a maximum of 6.5%.

    “we calculated an ejection fraction of 0.7% on average, with a maximum value of 6.5%”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    As hiPSC colonies expanded, space appeared to be created by ECM hydrolysis or by MMP-mediated ECM remodeling, and hiPSCs expressed MMPs 1, 5, and 6.

    “as colonies grew, it appeared that either ECM hydrolysis or direct remodeling of ECM via matrix metalloproteinases (MMPs) occurred to provide space, as hiPSCs expressed MMPs 1, 5, and 6”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Premisereasoning

    For cardiomyocyte differentiation, the bioink should ideally contain integrin-binding motifs that enhance cardiomyocyte-specific differentiation signaling.

    “To support differentiation, the bioink should ideally contain integrin-binding motifs that enhance signaling associated with cardiomyocyte-specific differentiation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisereasoning

    Transferring cardiomyocytes to a 3D printed structure requires disrupting prior cell-cell connections and using a cell strainer to remove aggregates, increasing the total cardiomyocytes needed.

    “This process requires the disruption of previously formed cell-cell connections, and a cell strainer is required to remove aggregated cardiomyocytes, which further adds to the total number of cardiomyocytes that must be produced prior to printing.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    3D bioprinting has been proposed to generate more geometrically complex tissues from the bottom-up.

    “3D bioprinting has been proposed as a means to generate more geometrically complex tissues from the bottom-up.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Early engineered heart tissues can generate force but do not pump fluid, limiting their utility for in vitro cardiac function and disease study.

    “At the same time, the lack of geometrical complexity limits the utility of the model for in vitro study of cardiac function and disease, as these tissues can generate force but do not pump fluid.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mesoderm specification has been linked to α5β1 integrin activation.

    “As one of many examples, mesoderm specification has been linked to α5β1 integrin activation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] source

  • May combine two claims.

    Study design and conductnone given

    hChaMPs were neither perfused in a bioreactor, exposed to controlled resistive mechanical stimulation, nor exposed to electrical stimulation.

    “hChaMPs were neither perfused in a bioreactor, exposed to controlled, resistive mechanical stimulation, nor exposed to electrical stimulation; all factors shown to improve maturation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Fibroblast-derived ECM engagement via β1, α2, and α3 integrins in human embryonic stem cells activates Wnt/β-catenin via MEK-ERK, driving endoderm differentiation.

    “In addition, engagement of fibroblast-derived ECM via β1, α2, and α3 integrins in human embryonic stem cells activates the Wnt/β-catenin pathway via the MEK-ERK pathway, which drives endoderm differentiation28.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] source

  • The quote only partly states this claim.

    Interpretationown data

    The greater RyR2 regularity suggests advanced maturation in the hChaMP.

    “suggestive of advanced maturation in the hChaMP.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Electrical stimulation can promote connectivity and improve the coordinated function of the hChaMP.

    “suggesting electrical stimulation can promote connectivity and further enhance the concerted function of the hChaMP.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The hChaMP stroke work and ejection fraction are comparable to existing systems.

    “Stroke work and ejection fraction are on par with existing systems”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The measured stroke volume is a little below the model value, probably because cardiomyocytes are unevenly distributed over the hChaMP surface.

    “The experimentally determined stroke volume is slightly lower than this, likely due to an uneven distribution of cardiomyocytes across the hChaMP surface.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The in silico model indicates that a thicker cell layer should raise the hChaMP volume output and improve pump function.

    “as our in silico model demonstrated, increasing the cell layer thickness should increase the volume output of the hChaMP, resulting in more robust pump function”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The macroscale beating outcome was enabled by an optimized bioink that supported extensive stem cell proliferation before differentiation, producing contiguous muscle walls up to 500 μm thick.

    “This important outcome was made possible by an optimized bioink that allowed extensive stem cell proliferation prior to differentiation to yield contiguous muscle walls of up to 500 μm in thickness.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationnone given

    The approach could be used for many other cell types that proliferate and migrate poorly after differentiation.

    “This approach could be applied to many other cell types with poor proliferative and migratory capacity following differentiation.”
    Quote in the paperNot a substantial claim
51 of 51

  • The paper relies on this without support.

    Cardiomyocytes do not proliferate or migrate readily.

    “More specifically, cardiomyocytes do not proliferate or migrate readily.”

  • The paper relies on this without support.

    A high cell density is required for the formation of functional cell-cell junctions.

    “it is challenging to achieve the high cell density required for the formation of functional cell-cell junctions while maintaining the structural support needed for an enclosed chamber.”

  • The authors state this as an assumption.

    The pipetted disk used for cell-density estimation has a thickness of 1 mm.

    “Assuming a disk thickness of 1 mm”

  • The authors state this as an assumption.

    The average cell contains 6 pg of DNA.

    “If the average cell contains 6 pg of DNA”

  • The authors state this as an assumption.

    In the in silico model, the entire wall thickness is occupied by cells.

    “1) the entirety of the wall thickness is occupied by cells”

  • The authors state this as an assumption.

    In the in silico model, about half of the wall thickness, 600–650 μm, is occupied by cells.

    “2) approximately half of the wall thickness (600–650 μm) is occupied by cells”

  • The authors state this as an assumption.

    In the in silico model, cellularization is confined to the outer 200–250 μm of the structure.

    “3) the cellularization is limited to the outer 200–250 μm of the structure”
7 of 7

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The mini-hearts demonstrated stroke works of ~0.10 -~0.18 μJ, which are the highest stroke work values compared to other engineered ventricles (6 nJ-57 nJ).

    “The mini-hearts demonstrated stroke works of ~0.10 -~0.18 µJ which, to our knowledge, are the highest stroke work values compared to other engineered ventricles (6 nJ-57 nJ) (Kupfer et al., 2020;R. A. Li et al., 2018;Macqueen et al., 2018).”

    From A human engineered mini-heart platform for mimicking ventricular pump function (2025) · cites this paper as Kupfer et al., 2020

    The Results section (Figure 7e) reports an average spontaneous stroke work of 14.5 ± 1.1 nJ, not approximately 0.10–0.18 µJ (100–180 nJ). The Discussion, under “Advances to the Field,” says stroke work is on par with existing systems, contrary to the claim that these mini-hearts had the highest values. (LLM, conf=0.93)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • Bridging length scales from the micro- and nanometer scale to the centimeter scale to induce intra- and intercellular organization within engineered heart models remains a major challenge in 3D printing functional organs and tissues.

    “As a result, bridging the length scales from the micro- and nanometer scale to the centimeter scale to induce both intra- and intercellular organization within engineered heart models remains a major challenge in 3D printing functional organs and tissues32, 34, 35.”

    From Fibre-infused gel scaffolds guide cardiomyocyte alignment in 3D-printed ventricles (2023) · cites this paper as [32]

    The Discussion describes the challenge of expanding microscale cardiac models to macroscale structures while preserving cell–cell interactions and geometric complexity. The Introduction likewise discusses difficulties forming functional cell–cell junctions in 3D-printed chambered models. The paper does not specifically frame this as bridging nanometer-to-centimeter scales to induce both intracellular and intercellular organization; page numbers are unavailable in our copy. (LLM, conf=0.83)

    Experimental — accuracy not yet measured

  • Simultaneous PV measurements are currently limited to invasive micro-catheter systems adapted from in vivo mouse experiments.

    “Simultaneous PV measurements are currently limited to invasive micro-catheter systems adapted from in vivo mouse experiments (Li et al., 2018; MacQueen et al., 2018; Kupfer et al., 2020; Mohammadi et al., 2022).”

    From Mini-hearts for disease modeling and drug testing – process optimization versus biological functionality (2026) · cites this paper as Kupfer et al., 2020

    The Results section reports that the authors inserted a conductance catheter with a pressure transducer into a chambered organoid and measured pressure and volume simultaneously. It does not say that simultaneous PV measurements are limited to such systems or that the catheter was adapted from in vivo mouse experiments. No page number is available in our copy. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • Existing models span a wide range, from approximately 174,000 cells per ECC to about 15 million cells, directly influencing both production cost and feasibility.

    “Existing models span a wide range - from approximately 174,000 cells per ECC to about 15 million cells - directly influencing both production cost and feasibility (Lee et al., 2008; Chang et al., 2022; Kupfer et al., 2020).”

    From Mini-hearts for disease modeling and drug testing – process optimization versus biological functionality (2026) · cites this paper as Kupfer et al., 2020

    The Discussion section on in situ differentiation (page unavailable) reports a starting concentration of 15 million cells per mL, not 15 million cells per model. The Introduction (page unavailable) says producing large numbers of cardiomyocytes is technically challenging, which supports a feasibility concern, but the paper does not establish the stated range of cells per model or its effect on production cost. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • More sophisticated fabrication strategies, including nanofabrication and 3D (bio)printing, offer greater control over geometry and microstructure but raise concerns about efficiency, cost, and throughput.

    “More sophisticated fabrication strategies, including nanofabrication and 3D (bio)printing, offer greater control over geometry and microstructure but raise concerns about efficiency, cost, and throughput (Lee et al., 2019; Kupfer et al., 2020; Esser et al., 2023).”

    From Mini-hearts for disease modeling and drug testing – process optimization versus biological functionality (2026) · cites this paper as Kupfer et al., 2020

    The Introduction and Results (page unavailable) support the geometry claim: the paper contrasts simple cast tissues with more complex 3D-bioprinted structures and reports high fidelity to a chambered digital template. It does not establish a general advantage in microstructural control or discuss nanofabrication, cost, or throughput. Its fabrication concerns instead include printability and cell viability, so those parts of the citing sentence are not supported by this source. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Optimized bioink names two different laminin components

    Minor

    The formulation description identifies laminin-111, whereas the later list of ingredients for the selected formulation identifies laminin-III.

  • Maximum muscle-wall thickness is stated inconsistently

    Minor

    The conclusion caps the muscle-wall thickness at 500 μm, but the results say the thickest region exceeded 500 μm.

2 of 2

Cites

Flagged first, then by citations

1 of the 64 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Kupfer, M. E., Lin, W.-H., Ravikumar, V., Qiu, K., Wang, L., Gao, L., Bhuiyan, D. B., Lenz, M., Ai, J., Mahutga, R. R., Townsend, D., Zhang, J., McAlpine, M. C., Tolkacheva, E. G., & Ogle, B. M.
Affiliations
  • University of Minnesota (US)
  • University of Alabama at Birmingham (US)
ORCID
Venue
Circulation Research
Publisher
Ovid Technologies (Wolters Kluwer Health)
Published
2020-07-03
Volume
127
Issue
2
Pages
207-224
ISSN
  • 0009-7330 (ISSN-L)
  • 1524-4571
DOI
10.1161/circresaha.119.316155
PMC
PMC8210857
OpenAlex
W3014021096
Cited by
329
References
64
Citations by year
2026: 45 · 2025: 55 · 2024: 63 · 2023: 51 · 2022: 58 · 2021: 47 · 2020: 10
FWCI
13.03
Type
journal article
Language
en
Open access
Locations
  • Circulation Research (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Heart, Lung, and Blood Institute (R01HL137204, R01HL149137, R01HL131017)
  • National Institute of General Medical Sciences (T32GM008347)
  • National Institute of Diabetes and Digestive and Kidney Diseases (P30DK079626)
  • National Institute of Biomedical Imaging and Bioengineering (DP2EB020537)
Topics
  • Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
Keywords
3D bioprinting, human induced pluripotent stem cells, cardiomyocyte differentiation, in situ differentiation, bioinks, extracellular matrix, electromechanical coupling, action potential propagation, cardiac tissue engineering, cardiac remodeling, tissue grafting
MeSH terms
Action Potentials, Cell Differentiation, Cells, Cultured, Humans, Myocardial Contraction, Organoids, Extracellular Matrix Proteins, Tissue Engineering, Myocytes, Cardiac, Cell Proliferation, Induced Pluripotent Stem Cells, Bioprinting

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read under definition version 3 (extract-v3) on 2026-09-28.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-09-28.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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