(486e) Production of Cardiac Tissue Spheroids from Encapsulated Hipscs Using a Novel Microfluidic System

Introduction: Cardiovascular disease is the leading cause of death
worldwide due in part to the inability for the heart to regenerate after damage
or disease. Current treatment methods are not sufficient at regenerating
damaged myocardium. However, tissue engineering holds promise for improving
cardiac function and structure. Engineered cardiac tissues can
be produced from cells combined with biomaterials for use in
drug-testing, studying development and disease mechanisms, and regenerative
medicine. In order for these treatments to transition to clinical use, large
numbers of CMs must be generated, as it is estimated
that billions of cells will be needed for a therapeutic effect. Because of
their highly specialized function and low turnover rate, native CMs cannot be
cultured long term in vitro.
Therefore, stem cell differentiation from human induced pluripotent stem cells
(hiPSCs) must be utilized. Typical protocols for generating engineered cardiac
tissue involve multiple cell-handling steps including differentiation of
hiPSCs, dissociation of the resulting CMs, followed by recombination with a
biomaterial. These numerous cell-handling steps can be detrimental to the CMs.
Previously in our lab, a single cell-handling approach was established where
hiPSCs were encapsulated within a biomaterial to form 3D microenvironment and
directly differentiated to produce cardiac tissue using the hybrid
biomaterials, PEG-fibrinogen (PEG-fb) and GelMA^1,2.
Although these tissues were successful in generating mature cardiac tissue,
this protocol is not amenable for scale-up and suspension culture. A spheroidal
platform is beneficial for large scale production of
cardiac tissue because of its ability to be used for suspension culture and for
direct injection. Building on our previous work, here, we present a rapid,
scalable, and single-cell handling approach to produce cardiac tissue spheroids
from encapsulated hiPSCs within a hybrid, photocrosslinkable biomaterial,
PEG-fb, using novel microfluidic system. This system
uses provides tight control over size and shape of the resulting spheroids and
has the potential to be leveraged in bioreactor-based culture for cell therapy.

Materials and Methods: This novel
microfluidic system uses a custom oil-and-water emulsion technique in which the
hiPSCs at a high concentration (25x10⁶ cells/mL) are
combined with the PEG-fb precursor solution. The PEG-fb precursor
solution containing the hiPSCs (discrete phase) is pumped in the top of the
PDMS device while mineral oil (continuous phase) is pumped through the bottom.
Spheroids are formed at the modified T-junction and travel through the outlet
channel where they are crosslinked using high intensity visible light and
collected for further experiments. Following encapsulation, the hiPSCs were
cultured for 3 days prior to initiation of cardiac differentiation which
follows the published protocols^3,4. The
size, area growth, and roundness were quantified using Image-J software. The
viability of the cells was assessed following
encapsulation using a Live/Dead Viability/Cytotoxicity Kit. Young’s Modulus was
determined using a Microsquisher parallel plate compression. The efficiency of
cardiac differentiation was quantified using flow cytometry in which the cells
were labelled with cardiac troponin T (cTnT, cardiac marker), Ki67
(proliferation marker), and P4HB (fibroblast marker). Temporal gene expression was assessed by isolating mRNA from the spheroids and
performing RT-qPCR. Assessment of CM distribution and morphology occurred
through immunostaining with cardiac markers (cTnT and aSA)
and gap junction protein marker, Cx43. Dissociated CMs
were seeded on a multielectrode array (MEA) and paced with electrical and
pharmacological stimuli.

Results and Discussion: This novel microfluidic system can be used to rapidly
produce highly uniform spheroids (75 min^-1)
with a crosslinking time of approximately 1 second. The spheroids were uniform
both within a batch and between batches with an average diameter of 900±40 mm and average roundness
of 0.96±0.02 (n=8 batches).
Encapsulation occurred on day -3 of cardiac differentiation, and the encapsulated
cells remained viable and continued to proliferate and grow within their 3D
microenvironment. Cardiac differentiation was initiated
on day 0, and spontaneous contractions were visualized by day 8 of cardiac
differentiation with synchronous contractions occurring by day 20. Engineered
cardiac spheroids exhibited efficient cardiac differentiation efficiency with
greater than 70% of the cells expressing cardiac marker, cTnT+. Appropriate
temporal changes in gene expression were quantified using RT-qPCR, including a
decrease in pluripotency gene, Oct4, as well as appropriate changes in cardiac
genes MLC2v, aMHC, and bMHC. Resulting CMs responded appropriately to
electrical and pharmacological stimuli when plated on the multielectrode array
(MEA). The spheroids exhibited 1:1 capture up to 6 Hz and responded
appropriately to b-adrenergic agonist
isoproterenol (Iso) and antagonist, propranolol
(Prop), indicating functionality of the CMs. Because of the high uniformity,
efficient cardiac differentiation, and appropriate gene expression and
functionality, these engineered cardiac microspheres have the potential to be used for cell production in a bioreactor, high-throughput
drug screening, and injectable cell therapy.

Conclusions:  Here, we present a novel
microfluidic system for cell encapsulation to directly
differentiate cardiac tissues. This system rapidly produces uniform
spheroids with high cardiac differentiation efficiency. The resulting CMs
exhibit appropriate responses to pharmaceutical and electrical stimuli, gene
expression, and cell staining markers. This technique has the potential to be
scaled-up for production in a bioreactor for cell therapy.

References:

1     Kerscher, P. et al. Direct hydrogel
encapsulation of pluripotent stem cells enables ontomimetic differentiation and
growth of engineered human heart tissues. Biomaterials 83, 383-395,
doi:10.1016/j.biomaterials.2015.12.011 (2016).

2     Kerscher, P. et al. Direct Production of Human Cardiac Tissues
by Pluripotent Stem Cell Encapsulation in Gelatin Methacryloyl. ACS
Biomaterials Science & Engineering 3, 1499-1509,
doi:10.1021/acsbiomaterials.6b00226 (2016).

3     Lian, X. et al. Directed cardiomyocyte
differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined
conditions. Nat Protoc 8, 162-175,
doi:10.1038/nprot.2012.150 (2013).

4     Burridge, P. W. et al. Chemically
defined generation of human cardiomyocytes. Nat Methods 11, 855-860,
doi:10.1038/nmeth.2999 (2014).