Chikahiro Imashiro

Bio: Chikahiro Imashiro is an academic researcher from Keio University. The author has contributed to research in topics: Cell culture & Trypsinization. The author has an hindex of 7, co-authored 22 publications receiving 137 citations.

Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves.

Abstract: Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process.

Fundamental Technologies and Recent Advances of Cell-Sheet-Based Tissue Engineering

Abstract: Tissue engineering has attracted significant attention since the 1980s, and the applications of tissue engineering have been expanding. To produce a cell-dense tissue, cell sheet technology has been studied as a promising strategy. Fundamental techniques involving tissue engineering are mainly introduced in this review. First, the technologies to fabricate a cell sheet were reviewed. Although temperature-responsive polymer-based technique was a trigger to establish and spread cell sheet technology, other methodologies for cell sheet fabrication have also been reported. Second, the methods to improve the function of the cell sheet were investigated. Adding electrical and mechanical stimulation on muscle-type cells, building 3D structures, and co-culturing with other cell species can be possible strategies for imitating the physiological situation under in vitro conditions, resulting in improved functions. Finally, culture methods to promote vasculogenesis in the layered cell sheets were introduced with in vivo, ex vivo, and in vitro bioreactors. We believe the present review that shows and compares the fundamental technologies and recent advances for cell-sheet-based tissue engineering should promote further development of tissue engineering. The development of cell sheet technology should promote many bioengineering applications.

Enzyme-free cell detachment mediated by resonance vibration with temperature modulation.

Abstract: Cell detachment is an essential process in adherent cell culture. However, trypsinization, which is the most popular detachment technique used in culture, damages cellular membranes. Reducing cellular membrane damage during detachment should improve the quality of cell culture. In this article, we propose an enzyme-free cell detachment method based on resonance vibration with temperature modulation. We developed a culture device that can excite a resonance vibration and control temperature. We then evaluated the cell detachment ratio and the growth response, observed the morphology, and analyzed the cellular protein of the collected cells-mouse myoblast cell line (C2C12). With the temperature of 10°C and the maximum vibration amplitude of 2 μm, 77.9% of cells in number were successfully detached compared with traditional trypsinization. The 72-h proliferation ratio of the reseeded cells was similar to that with trypsinization, whereas the proliferation ratio of proposed method was 12.6% greater than that of trypsinization after freezing and thawing. Moreover, the cells can be collected relatively intact and both intracellular and cell surface proteins in the proposed method were less damaged than in trypsinization. These results show that this method has definite advantages over trypsinization, which indicates that it could be applied to subcultures of cells that are more susceptible to trypsin damage for mass culture of sustainable clinical use. Biotechnol. Bioeng. 2017;114: 2279-2288. © 2017 Wiley Periodicals, Inc.

Detachment of cell sheets from clinically ubiquitous cell culture vessels by ultrasonic vibration.

Abstract: Proteinases that digest the extracellular matrix are usually used to harvest cells from culture vessels in a general culture process, which lowers the initial adhesion rate in regenerative medicine. Cell sheet engineering is one of the most important technologies in this field, especially for transplantation, because fabricated cell sheets have rich extracellular matrixes providing strong initial adhesion. Current cell sheet fabrication relies on temperature-responsive polymer-coated dishes. Cells are cultured on such specialized dishes and subjected to low temperature. Thus, we developed a simple but versatile cell sheet fabrication method using ubiquitous culture dishes/flasks without any coating or temperature modulation. Confluent mouse myoblasts (C2C12 cell line) were exposed to ultrasonic vibration from underneath and detached as cell sheets from entire culture surfaces. Because of the absence of low temperature, cell metabolism was statically increased compared with the conventional method. Furthermore, viability, morphology, protein expression, and mRNA expression were normal. These analyses indicated no side effects of ultrasonic vibration exposure. Therefore, this novel method may become the standard for cell sheet fabrication. Our method can be easily conducted following a general culture procedure with a typical dish/flask, making cell sheets more accessible to medical experts.

Focused surface acoustic wave locally removes cells from culture surface.

Abstract: Regenerative medicine and drug development require large numbers of high-quality cells, usually delivered from in vitro culturing. During culturing, the appearance of unwanted cells and an inability to remove them without damaging or losing most if not all the surrounding cells in the culture reduce the overall quality of the cultured cells. This is a key problem in cell culturing, as is the inability to sample cells from a culture as desired to verify the quality of the culture. Here, we report a method to locally remove cells from an adherent cell culture using a 100.4 MHz focused surface acoustic wave (SAW) device. After exposing a plated C2C12 mouse myoblast cell culture to phosphate buffered solution (PBS), ultrasound from the SAW device transmitted into the cell culture via a coupling water droplet serves to detach a small grouping of cells. The cells are removed from an area 6 × 10-3 mm2, equivalent to about 12 cells, using a SAW device-Petri dish water gap of 1.5 mm, a PBS immersion time of 300 s, and an input voltage of 75 V to the SAW device. Cells were released as desired 90% of the time, releasing the cells from the target area nine times out of ten runs. In the one trial in ten that fails, the cells partially release and remain attached due to inter-cellular binding. By making it possible to target and remove small groups of cells as desired, the quality of cell culturing may be significantly improved. The small group of cells may be considered a colony of iPS cells. This targeted cell removal method may facilitate sustainable, contamination-free, and automated refinement of cultured cells.

Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves.

Abstract: Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process.

The waves that make the pattern: a review on acoustic manipulation in biomedical research.

Abstract: Novel approaches, combining technology, biomaterial design, and cutting-edge cell culture, have been increasingly considered to advance the field of tissue engineering and regenerative medicine. Within this context, acoustic manipulation to remotely control spatial cellular organization within a carrier matrix has arisen as a particularly promising method during the last decade. Acoustic or sound-induced manipulation takes advantage of hydrodynamic forces exerted on systems of particles within a liquid medium by standing waves. Inorganic or organic particles, cells, or organoids assemble within the nodes of the standing wave, creating distinct patterns in response to the applied frequency and amplitude. Acoustic manipulation has advanced from micro- or nanoparticle arrangement in 2D to the assembly of multiple cell types or organoids into highly complex in vitro tissues. In this review, we discuss the past research achievements in the field of acoustic manipulation with particular emphasis on biomedical application. We survey microfluidic, open chamber, and high throughput devices for their applicability to arrange non-living and living units in buffer or hydrogels. We also investigate the challenges arising from different methods, and their prospects to gain a deeper understanding of in vitro tissue formation and application in the field of biomedical engineering.

High Frequency Sonoprocessing: A New Field of Cavitation-Free Acoustic Materials Synthesis, Processing, and Manipulation.

Abstract: Ultrasound constitutes a powerful means for materials processing. Similarly, a new field has emerged demonstrating the possibility for harnessing sound energy sources at considerably higher frequencies (10 MHz to 1 GHz) compared to conventional ultrasound (⩽3 MHz) for synthesizing and manipulating a variety of bulk, nanoscale, and biological materials. At these frequencies and the typical acoustic intensities employed, cavitation-which underpins most sonochemical or, more broadly, ultrasound-mediated processes-is largely absent, suggesting that altogether fundamentally different mechanisms are at play. Examples include the crystallization of novel morphologies or highly oriented structures; exfoliation of 2D quantum dots and nanosheets; polymer nanoparticle synthesis and encapsulation; and the possibility for manipulating the bandgap of 2D semiconducting materials or the lipid structure that makes up the cell membrane, the latter resulting in the ability to enhance intracellular molecular uptake. These fascinating examples reveal how the highly nonlinear electromechanical coupling associated with such high-frequency surface vibration gives rise to a variety of static and dynamic charge generation and transfer effects, in addition to molecular ordering, polarization, and assembly-remarkably, given the vast dimensional separation between the acoustic wavelength and characteristic molecular length scales, or between the MHz-order excitation frequencies and typical THz-order molecular vibration frequencies.

Fundamental Technologies and Recent Advances of Cell-Sheet-Based Tissue Engineering

Abstract: Tissue engineering has attracted significant attention since the 1980s, and the applications of tissue engineering have been expanding. To produce a cell-dense tissue, cell sheet technology has been studied as a promising strategy. Fundamental techniques involving tissue engineering are mainly introduced in this review. First, the technologies to fabricate a cell sheet were reviewed. Although temperature-responsive polymer-based technique was a trigger to establish and spread cell sheet technology, other methodologies for cell sheet fabrication have also been reported. Second, the methods to improve the function of the cell sheet were investigated. Adding electrical and mechanical stimulation on muscle-type cells, building 3D structures, and co-culturing with other cell species can be possible strategies for imitating the physiological situation under in vitro conditions, resulting in improved functions. Finally, culture methods to promote vasculogenesis in the layered cell sheets were introduced with in vivo, ex vivo, and in vitro bioreactors. We believe the present review that shows and compares the fundamental technologies and recent advances for cell-sheet-based tissue engineering should promote further development of tissue engineering. The development of cell sheet technology should promote many bioengineering applications.

The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia

Abstract: Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine–creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine–creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens.