Imaging cell picker: A morphology-based automated cell separation system on a photodegradable hydrogel culture platform

Event Abstract Back to Event Development of photodegradable gelatin hydrogels and image analysis technique for automatic optical cell separation system based on the cellular morphology in embedding culture Masato Tamura1, Shinji Sugiura1, Taku Satoh1, Toshiyuki Kanamori1, Shibuta Mayu2, Kei Kanie2, Ryuji Kato2, Hirofumi Matsui3 and Masumi Yanagisawa4 1 National Institute of Advanced Industrial Science and Technology (AIST), Department of Life Science and Biotechnology, Japan 2 Nagoya University, Graduate School of Pharmaceutical Sciences, Japan 3 University of Tsukuba, Faculty of Medicine, Japan 4 Engineering System Co., Ltd., Japan This paper reports novel cell separation strategy and automated cell separation system based on the cellular morphology under 3D culture environment in the photodegradable hydrogel. Recently, we developed gelatin-based photodegradable hydrogels by NHS-activated-ester reaction [1], and applied this hydrogels to optical cell separation [2]. More recently, we developed “click-crosslinkable and photodegradable gelatin hydrogels” [3]. The present study applied this hydrogels to the most promising application, cell separation based on the cellular morphology. The present study includes proof concept of morphology-based cell separation and development of automatic cell separation system. 3D culture environment with a specific extracellular matrix regulates cellular function and phenotype. In addition, cancer cell morphology is alternated depending on its malignancy in 3D culture environment [4]. Recently we developed the predication model of stem cell differentiation by image analysis [5]. In this paper, we introduced this image analysis technique and the features of click-crosslinkable and photodegradable gelatin hydrogels to the automated morphology-based cell separation system in 3D culture environment in the above mentioned photodegradable gelatin hydrogels. Figure 1 shows the schematic procedure for optical cell separation based on the cellular morphology under 3D culture environment in the photodegradable hydrogel. Suspension of cells including heterogeneous population is mixed with pregel solutions and cells are encapsulated in the gelatin-based photodegradable hydrogels (Figure 1a). After the culture in 3D environment (Figure 1b), microscopic images of the cells are captured. The captured images are analyzed to distinguish the target cells from the other cells by using the image analysis algorithm, which we previously developed for analyzing stem cells (Figure 2) [5]. The hydrogels around the target area is irradiated with UV light (Figure 1c). The cells in the irradiated area are collected by automated pipetting system (Figure 1d). We developed automated system for this optical cell separation procedure, including cultivation, image acquisition, image analysis, light irradiation, and pipetting for cell collection (Figure 3). We are currently developing an automated image analysis algorithm to distinguish cancer cells from normal cells under 3D environment. The automated optical cell separation system with image analysis algorithm will be applied to the establishment of novel cancer-cell lines from clinical samples such as biopsy tissue. KAKENHI (14J07186); NEDO (1009004)

The present study includes proof concept of morphology-based cell separation and development of automatic cell separation system. 3D culture environment with a specific extracellular matrix regulates cellular function and phenotype. In addition, cancer cell morphology changes depending on its malignancy in 3D culture environment. The cell separation system in 3D culture environment should need to obtain the cells according to its morphology, which includes cell phenotypes. Recently, we developed gelatin-based photodegradable hydrogels, and applied this hydrogels to optical cell separation. The target cells in the photodegradable hydrogels were successfully separated by the optical cell separation, the separated cells was growth on another dish. On the other hand, we recently developed the predication model of stem cell differentiation by image analysis. The image analysis technique and the photodegradable gelatin hydrogels are included in the automated morphology-based cell separation system in the 3D culture environment. For forming cell encapsulated-photodegradable hydrogels, suspension of cells including heterogeneous population is mixed with pregel solutions and cells are encapsulated in the gelatin-based photodegradable hydrogels. After the culture in 3D environment, microscopic images of the cells are captured. The captured images are analyzed to distinguish the target cells from the other cells by using the image analysis algorithm, which we previously developed for analyzing stem cells. The hydrogels around the target area is irradiated with light (365nm). The cells in the irradiated area are collected by automated pipetting system. We developed automated system for this optical cell separation procedure, including cultivation, image acquisition, image analysis, light irradiation, and pipetting for cell collection. We demonstrated automated optical cell separation using the model culture system. Normal gastric mucosal cells were cultured in the photodegradable hydrogels. After cultivation for 1 week, the cells were irradiated the light for 5 to 20 min. The cells in the irradiated area were collected by automated pipetting and transferred into a collection dish. The collected cells were viable and attached in the collection dish after collection. We are currently developing an automated image analysis algorithm to distinguish cancer cells from normal cells under 3D environment. The automated optical cell separation system with image analysis algorithm will be applied to the establishment of novel cancer-cell lines from clinical samples such as biopsy tissue. Citation Format: Hirofumi Matsui, Shinji Sugiura, Masato Tamura, Toshiyuki Kanamori, Toshiyuki Takagi, Taku Satou, Ryuji Kato, Kei Kanie, Mayu Shibuta. Development of cellular morphology-based separation system for three-dimensional culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5788. doi:10.1158/1538-7445.AM2017-5788

Continuous flow microfluidic separation and processing of rare cells and bioparticles found in blood – A review

Development of genetic profiles from the biological mixtures has remained challenging, although modern-day technologies may help forensic scientists to attain a reliable genetic profile in the identification of the accused. In the case of rape, vaginal swab exhibits usually contain epithelial cells of victims and sperm cells of accused, such samples are more challenging when there is more than one contributor. In such cases, separation of distinct cells from a mixture that includes blood cells, epithelial cells and sperm cells for their single genetic profile is important. In the last ten decades several new techniques were developed and invented for the separation of single cell from the biological mixture that includes differential lysis, laser micro-dissection, cell sorting (FACS), sieve-based filtration, (vi) micro-fluidic devices or immunomagnetic beads cell separation of fresh samples, and the magnetic activated cell sorting (MACS). Out of them, some techniques have been commonly applied for cell separation in forensic biology. Each technique has its own limitation. Some recent studies showed, magnetic activated cell sorting (MACS), laser capture microdissection (LCM), DEPArray technology and fluorescence activated cell sorting (FACS) has proved to be effective in separation of single cell from cell mixtures. Therefore, in this review we have evaluated these four alternative methods and their potential application in the modern-day over the others for the separation of a single cell from the mixture. In this review we also discuss the advantage of these methods and their modern–day applicability and acceptance in the forensic world.

Cell separation is a critical process in biological and biomedical research, and in clinical therapy. The gold standard of cell separation techniques are fluorescence-activated cell sorting (FACS) and magneticactivated cell sorting (MACS). Despite this, there are still some limitations with FACS and MACS such as low recovery and purity, and affecting the cell physiology and viability. Herein, we report a universal cell separation approach based on reversible binding between N3′-ethyl biotin and streptavidin, which allows for fast capturing of target cells from cell mixture, and fast and complete releasing cells from magnetic beads under mild, physiological conditions. This universal method for detaching cells from magnetic beads provides an efficient cell separation technique based on MACS to give high recovery, high purity, and high viability of cells.

Purification of human iPSC-derived cells at large scale using microRNA switch and magnetic-activated cell sorting.

Cell sorting is an essential and efficient experimental tool for the isolation and characterization of target cells. A three-dimensional environment is crucial in determining cell behavior and cell fate in biological analysis. Herein, we have applied photodegradable hydrogels to optical cell separation from a 3D environment using a computer-controlled light irradiation system. The hydrogel is composed of photocleavable tetra-arm polyethylene glycol and gelatin, which optimized cytocompatibility to adjust a composition of crosslinker and gelatin. Local light irradiation could degrade the hydrogel corresponding to the micropattern image designed on a laptop; minimum resolution of photodegradation was estimated at 20 µm. Light irradiation separated an encapsulated fluorescent microbead without any contamination of neighbor beads, even at multiple targets. Upon selective separation of target cells in the hydrogels, the separated cells have grown on another dish, resulting in pure culture. Cell encapsulation, light irradiation and degradation products exhibited negligible cytotoxicity in overall process.

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS+) and STRO-1-negative (MACS−) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS+ and MACS− cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS+ and MACS− cell fractions showed plastic adherence. MACS+ cells, in contrast to MACS− cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS+ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS− cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS+ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS− cells demonstrated slight osteogenic potential. Unstimulated MACS+ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS− cells demonstrated higher expression of osteonectin (P<0.05; Mann–Whitney). The present study is the first to compare gingival MACS+ and MACS− cell populations demonstrating that MACS+ cells, in contrast to MACS− cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS+ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS+ cells are a unique renewable source of multipotent stem/progenitor cells.

Living cells in human body exhibit their function in microstructured three dimensional (3D) environments composed of soluble factors, extracellular matrix (ECM), and neighboring cells. Fabricating biological 3D in vitro models of tissue and organ is a critical step for developing new strategies for cell based assay in drug discovery. To date, a number of research groups have developed micropatterning of hydrogels using photocurable and photodegradable hydrogels. This chapter reviews recent development on the techniques for microscale 3D fabrication of biomaterials by means of photolithography. Also, this chapter introduces an activated-ester-type photocleavable crosslinker, which we recently synthesized to generate photodegradable hydrogels using biocompatible materials such as polyethylene glycol and gelatin. This new type of crosslinker enabled convenient preparation of photodegradable hydrogel by two component mixing reaction. The hydrogels were degraded by micropatterned light irradiation, local light irradiation, and two-photon excitation. This simple and convenient approach to prepare and fabricate photodegradable hydrogels is creating new opportunity for novel cell manipulation and 3D tissue engineering techniques.

A Key Role for CC Chemokine Receptor 1 in T-Cell-Mediated Respiratory Inflammation

Germ line or hypothalamus-specific deletion of Y2 receptors in mice results in a doubling of trabecular bone volume. However, the specific mechanism by which deletion of Y2 receptors increases bone mass has not yet been identified. Here we show that cultured adherent bone marrow stromal cells from Y2(-/-) mice also demonstrate increased mineralization in vitro. Isolation of two populations of progenitor cell types, an immature mesenchymal stem cell population and a more highly differentiated population of progenitor cells, revealed a greater number of the progenitor cells within the bone of Y2(-/-) mice. Analysis of Y receptor transcripts in cultured stromal cells from wild-type mice revealed high levels of Y1 but not Y2, Y4, Y5, or y6 receptor mRNA. Interestingly, germ line Y2 receptor deletion causes Y1 receptor down-regulation in stromal cells and bone tissue possibly due to the lack of feedback inhibition of NPY release and subsequent overstimulation of Y1 receptors. Furthermore, deletion of Y1 receptors resulted in increased bone mineral density in mice. Together, these findings indicate that the greater number of mesenchymal progenitors and the altered Y1 receptor expression within bone cells in the absence of Y2 receptors are a likely mechanism for the greater bone mineralization in vivo and in vitro, opening up potential new treatment avenues for osteoporosis.

Recent advances in science and technology have led to revolutions in many scientific and industrial fields. The term lab on a chip, or in other words, performing a variety of complex analyses in just a short time and a minimal space, is a term that has become very common in recent years, and what used to be a dream has now come to life in practice. In this paper, we tried to investigate a specific type of lab technology on a chip, which is, of course, one of the most common, namely the knowledge and technology of cell separation by using a microfluidic technique that can be separated based on size and deformation, adhesion and electrical properties. The tissue of the human body is degraded due to injury or aging. It is often tried to treat this tissue disorder by using drugs, but they are not always enough. Stem cell-based medicine is a novel form that promises the restoration or regeneration of tissues and functioning organs. Although many models of microfluidic systems have been designed for cell separation, choosing the appropriate device to achieve a reliable result is a challenge. Therefore, in this study, Fluorescence Activated Cell Sorting (FACS), Dielectrophoresis (DEP), Magnetic Activated Cell Sorting (MACS), and Acoustic microfluidic system are four distinct categories of active microfluidic systems explored. Also, the advantages, disadvantages, and the current status of the devices mentioned in these methods are reviewed.

Objective To compare effect of chemotherapy agent DDP to MACS in sorting cancer stemcells (CSC) of laryngeal carcinoma cell line Hep-2. Methods CD133 magnetic beads were applied to sort Hep-2 cells. Different dosages of DDP were used to treat Hep-2 cells for 48 hours. Enrichment rate of CD133+ cells by MACS and after DDP treatment was detected by Flow Cytometer (FCM). Morphologic change was observed under inverse-phase microscope. Results FCM showed that the sorting rate of CD133+ cells through MACS was 64.33 %, while after DDP treatment for 48 hours, the rate of CD133+ cells was enriched significantly in each dosage of DDP, with the maximal rate was 50.7 %, in the dosage of 4 μg/ml. There was a significantly difference between MACS and each of DDP group (P <0.01). Cells treated with DDP were abnormal in morphology. Conclusion MACS and DDP sorting has respective advantages in enriching CSC in Hep-2 cell lines. Key words: Cancer stem cell (CSC); Enrich; Magnetic activated cell sorting (MACS); Chemotherapy agents; CD133

Methodological comparison of FACS and MACS isolation of enriched microglia and astrocytes from mouse brain

：Objective To establish aeconomic and stable method to induce and culture dendritic cells (DCs) from peripheralblood of human being, and compare with the magnetic activated cell sorting. MethodsMonocytes were isolated from health donors peripheral blood mononuclear cells(PBMC) bydensity gradient separation,cultured and compared with that of cells isolated by themagnetic activated cell sorting or adherent culture,respectively. PBMC were cultured withrecombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) andrecombinant human interleukin-4(rhIL-4) for 6 days to induce the growth of DCs.Morphological changes was observed under inverted microscope. Meanwhile, cell viabilitywas tested at the 3rd, 5th, 6th day,respectively. The phenotypes, like CD14, CDla, HLA-DRwere analyzed with flow cytometry after PBMC were adherent cultured for 1, 2, 5 h. Afteradding human recombinant cytokines, the phenotypes of acquired cells surface markers,CD14, CD1a, CD86, CD83 and HLA-DR would be detected and compared with flow cytometry. Tcells proliferating activity was determined by allogeneic mixed lymphocyte reaction invitro. Results After adherent culture for 2 h, the acquired DCs showed typical morphology.Cell viability was decreased at days 5th, 6th[(53.333 ±5.774)%,(38.333 ± 7.638)%] thanthat at day of 3rd[(68.667 ± 3.215)%, all P ＜0.05] with themagnetic activated cell sorting, but with adherent culture method, the difference was notstatistically significant (F = 0.737,P＞0.05) at days of3rd, 5th, 6th[(92.667 ± 3.055)%,(94.000 ± 1.000)%,(94.667 ± 1.528)%]. Moreover,comparedwith the magnetic activated cell sorting, there were differences in cell viability ofadherent culture method at days of 3rd, 5th, 6th(t = 9.374, 12.021,12.527, all P ＜ 0.05). Before and after using the magnetic activated cell sorting,the expression of CD14 were (32.457 ± 12.351) %, (41.914 ± 14.858)%, respectively. Thedifference was not statistically significant(t = 1.295, P ＞ 0.05). After culturing for 2 h, the expression of CD14[(35.267 ± 4.658)%]washigher than those of culturing for 1, 5 h[(15.033 ± 6.189)%, (21.233 ± 4.895)%, all P＜ 0.05]. Compared with the 1st day[(32.328 ±14.517)%], the CD14 expression level[(2.200 ± 1.356)%] on surface of DCs wassignificantly reduced(t = 5.467, P ＜ 0.05) at the 6th day ofculturing, the CD1a expression level[(43.371 ±16.250)%] was remarkablely increased thanthat of the 1st day[(12.300 ± 6.223)%, t = 2.545, P ＜ 0.05];while the expressions of CD86, CD83, HLA-DR[(16.857 ± 5.686)%,(9.343 ±5.230)%,(72.800 ± 17.881)%] were similar(t = 0.652,1.137,0.907, all P ＞ 0.05) compared with that of the 1st day[(12.550 ± 16.758)%, (6.250±1.323)%, (64.671 ± 15.588)%]. In mixed lymphocytes reactions, with increasing oflymphocytes, T lymphocytes proliferating activities were reduced. In the magneticactivated cell sorting, when the ratio of DCs and lymphocytes were 1: 50, 1: 100, cellsproliferation ability(1.502 ± 0.055,1.507 ± 0.029) were lower than that of ratio of 1:10(1.859 ± 0.049, all P ＜ 0.05);in adherent culturemethod, the ratio of DCs and lymphocytes was 1: 100, the cells proliferation ability(1.545± 0.066) was decreased than that of ratio 1: 10(2.015 ± 0.301, P ＜ 0.05). When the proportion of DCs and lymphocytes remained thesame, the capacity to stimulate T lymphocyte was similar of the two methods(P ＞ 0.05). Conclusions Compariedwith the magnetic activated cell sorting, after culture of PBMC for 2 h the induction ofDCs can produce better formed and functional cells, and this method is stable,simple,economic, and is a suitable method for basic and clinical research of DCs in vitro. Key words: Dendritic cells; Cell culture techniques; Cytokines; Flowcytometry; Lymphocyte culture test,mixed