Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation

Intracellular compartments are functional units that support the metabolic processes within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer customised complex emulsion droplets as a multicompartment artificial cell chassis, using multiphase microfluidics and acoustic levitation. Such levitated constructs provide free-standing, dynamic, definable droplet networks for the encapsulation and organisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and large-conductance mechanosensitive channel (MscL) and their activation. The assembly of droplet networks is three-dimensionally patterned with fluidic inputs configurations determining droplet contents and connectivity. Whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. In addition, a mechanosensitive channel, MscL, can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact control of membrane protein function. Collectively, this will expand our capability to program and operate increasingly sophisticated artificial cells as life-like materials.

Reversable deformation of artificial cell colony for muscle behavior mimicry triggered by actin polymerization

The mimicry of living tissues from artificial cells is beneficial to understanding the interaction mechanism among cells, as well as holding great potentials in the tissue engineering field. Self-powered artificial cells capable of reversible deformation are developed by encapsulating living mitochondria, actin proteins, and methylcellulose. Upon the addition of pyruvate molecules, the mitochondria produce ATP molecules as energy sources to trigger the polymerization of actin. ATP molecules were produced by mitochondria (2.76×1010/ml) with the concentrations of 35.8±3.2 µ M, 158.2±19.3 µ M and 200.7±20.1 µ M by adding pyruvate molecules with the concentration of 3 µ M; 12 µ M and 21 µ M, respectively. The reversible deformation of artificial cells is experienced with spindle shape resulting from the polymerization of actins to form filaments adjacent to the lipid bilayer, subsequently back to spherical shape resulting from the depolymerization of actin filaments upon laser irradiations. The linear colonies composed of these artificial cells exhibit collective contraction and relaxation behavior to mimic muscle tissues. At the stage of maximum contraction, the long axis of each GUV is in parallel to each other. All colonies are synchronized in the contraction phase. The deformation of each GUV in the colonies is influenced by its adjacent GUVs. The muscle-like artificial cell colonies paved the path to develop sustainably self-powered artificial tissues in the field of tissue engineering.

CBMS-1 Targeting amino acid metabolic vulnerabilities in IDH-mutant and IDH-wildtype gliomas

IDH-wildtype glioma and IDH-mutant glioma have different genetical and metabolic background although their histological appearances are similar. To reveal the difference in metabolites between IDH-wildtype and IDH-mutant glioma, and to find the effective treatment targeting cancer metabolism according to the status of IDH in gliomas, two artificial cell lines made from normal human astrocyte, NHAE6E7hTERTRas (IDH-wildtype) and NHAE6E7hTERTIDHmut (IDH-mutant), were investigated. RNA-seq analysis revealed that about 10% of changed genes were involved with metabolism. Capillary electrophoresis- and ion chromatography-coupled mass spectrometry revealed that the amount of asparagine was lower in NHAE6E7hTERTRas cells compared with NHAE6E7hTERTIDHmut cells. L-asparaginase, which converts asparagine into aspartate, was more effective in former cells. L-asparaginase induced autophagy and inhibition of autophagy by 3-MA suppressed L-asparaginase-induced antitumor effect. Adding asparagine into the culture medium rescued the antitumor effect of L-asparaginase. L-asparaginase increased the expression of asparagine synthetase (ASNS) and inhibition of ASNS enhanced the antitumor effect of L-asparaginase. Metabolic assay also showed the lower amount of glutamine, glutamate and 2-oxoglutarate in NHAE6E7hTERTIDHmut cells than NHAE6E7hTERTRas cells. Inhibition of GLUD1 which converts glutamate to 2-oxoglutarate, suppressed proliferation of the cells by inducing ROS and apoptosis in NHAE6E7hTERTIDHmut cells. Exogeneous dimethyl 2-oxoglutarate rescued the cytotoxicity by GLUD1 inhibitor, suggesting decreased 2-oxoglutarate was associated with GLUD1 inhibitor-induced cytotoxicity. ROS inhibitor, NAC suppressed GLUD1 inhibitor-induced ROS, apoptosis, and cytotoxicity in NHAE6E7hTERTIDHmut cells, revealing that cytotoxicity by GLUD1 inhibitor was at least partially due to the inhibitor-induced ROS. Other IDH-wildtype glioma cells, U251 and U87 showed similar sensitivity to L-asparaginase and GLUD1 inhibitor to NHAE6E7hTERTRas, whereas U251 expressing mutant IDH1 showed similar sensitivity to GLUD1 inhibitor to NHAE6E7hTERTIDHmut, which suggested that the difference of sensitivity to each reagent was due to the status of mutant IDH. L-asparaginase and GLUD1 inhibitor will be new therapeutic options for IDH-wildtype glioma and IDH-mutant glioma, respectively.

Artificial Cell Encapsulation for Biomaterials and Tissue Bio-Nanoengineering: History, Achievements, Limitations, and Future Work for Potential Clinical Applications and Transplantation

Pancreatic β-cell loss and failure with subsequent deficiency of insulin production is the hallmark of type 1 diabetes (T1D) and late-stage type 2 diabetes (T2D). Despite the availability of parental insulin, serious complications of both types are profound and endemic. One approach to therapy and a potential cure is the immunoisolation of β cells via artificial cell microencapsulation (ACM), with ongoing promising results in human and animal studies that do not depend on immunosuppressive regimens. However, significant challenges remain in the formulation and delivery platforms and potential immunogenicity issues. Additionally, the level of impact on key metabolic and disease biomarkers and long-term benefits from human and animal studies stemming from the encapsulation and delivery of these cells is a subject of continuing debate. The purpose of this review is to summarise key advances in this field of islet transplantation using ACM and to explore future strategies, limitations, and hurdles as well as upcoming developments utilising bioengineering and current clinical trials.

Modularize and Unite: Toward Creating a Functional Artificial Cell

An artificial cell is a simplified model of a living system, bringing breakthroughs into both basic life science and applied research. The bottom-up strategy instructs the construction of an artificial cell from nonliving materials, which could be complicated and interdisciplinary considering the inherent complexity of living cells. Although significant progress has been achieved in the past 2 decades, the area is still facing some problems, such as poor compatibility with complex bio-systems, instability, and low standardization of the construction method. In this review, we propose creating artificial cells through the integration of different functional modules. Furthermore, we divide the function requirements of an artificial cell into four essential parts (metabolism, energy supplement, proliferation, and communication) and discuss the present researches. Then we propose that the compartment and the reestablishment of the communication system would be essential for the reasonable integration of functional modules. Although enormous challenges remain, the modular construction would facilitate the simplification and standardization of an artificial cell toward a natural living system. This function-based strategy would also broaden the application of artificial cells and represent the steps of imitating and surpassing nature.

TAMI-56. TARGETING AMINO ACID METABOLIC VULNERABILITIES IN IDH-MUTANT AND IDH-WILDTYPE GLIOMAS

IDH-wildtype glioma and IDH-mutant glioma have different genetical and metabolic background although their histological appearances are similar. The aim of the study is to reveal the difference in metabolites between IDH-wildtype glioma and IDH-mutant glioma, and to find the effective treatment targeting cancer metabolism according to the status of IDH in gliomas. Two artificial cell lines made from normal human astrocyte were used: NHAE6E7hTERTRas (IDH-wildtype) and NHAE6E7hTERTIDHmut (IDH-mutant). Capillary electrophoresis time-of-flight-mass spectrometry (CE-TOFMS) revealed that the amount of asparagine was lower in NHAE6E7hTERTRas cells compared with NHAE6E7hTERTIDHmut cells. L-asparaginase, which converts asparagine into aspartate, was more effective in the former cells than the latter cells. L-asparaginase induced autophagy and inhibition of autophagy by 3-MA suppressed L-asparaginase-induced antitumor effect. Adding asparagine into the culture medium rescued the antitumor effect of L-asparaginase. L-asparaginase increased the expression of asparagine synthetase (ASNS) and genetical or pharmacological inhibition of ASNS enhanced the antitumor effect of L-asparaginase. CE-TOFMS showed the lower amount of glutamine, glutamate and 2-oxoglutarate in NHAE6E7hTERTIDHmut cells than NHAE6E7hTERTRas cells. GLUD1 inhibitor inhibited proliferation by inducing higher ROS level and apoptosis in NHAE6E7hTERTIDHmut cells than NHAE6E7hTERTRas cells. ROS inhibitor, NAC suppressed GLUD1 inhibitor-induced ROS, apoptosis, and cytotoxicity in NHAE6E7hTERTIDHmut cells. Exogeneous dimethyl 2-oxoglutarate rescued the cytotoxicity induced by GLUD1 inhibitor. L-asparaginase and GLUD1 inhibitor will be new therapeutic option for IDH-wildtype glioma and IDH-mutant glioma, respectively.

Morphological Fabrication of Rubber Cutaneous Receptors Embedded in a Stretchable Skin-Mimicking Human Tissue by the Utilization of Hybrid Fluid

Sensors are essential in the haptic technology of soft robotics, which includes the technology of humanoids. Haptic sensors can be simulated by the mimetic organ of perceptual cells in the human body. However, there has been little research on the morphological fabrication of cutaneous receptors embedded in a human skin tissue utilizing artificial materials. In the present study, we fabricated artificial, cell-like cutaneous receptors embedded in skin tissue mimicking human skin structure by utilizing rubber. We addressed the fabrication of five cutaneous receptors (free nerve endings, Krause and bulbs, Meissner corpuscles, Pacinian corpuscles and Ruffini endings). In addition, we investigated the effectiveness of the fabricated tissue for mechanical and thermal sensing. At first, in the production of integrated artificial skin tissue, we proposed a novel magnetic, responsive, intelligent, hybrid fluid (HF), which is suitable for developing the hybrid rubber skin. Secondly, we presented the fabrication by utilizing not only the HF rubber but our previously proposed rubber vulcanization and adhesion techniques with electrolytic polymerization. Thirdly, we conducted a mechanical and thermal sensing touch experiment with the finger. As a result, it demonstrated that intelligence as a mechanoreceptor or thermoreceptor depends on its fabric: the HF rubber sensor mimicked Krause and bulbs has the thermal and pressing sensibility, and the one mimicked Ruffini endings the shearing sensibility.