MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

In Vitro Invasive Pattern of Hepatocellular Carcinoma Cell Line HCCLM9 Based on Three-dimensional Cell Culture and Quantum Dots Molecular Imaging

Summary： This study aimed to establish a new in vitro three-dimensional （3D） cell culture and use quantum dots （QDs） molecular imaging to examine the invasive behaviors of hepatocellular carcinoma （HCC） cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with se- rum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morpho- logical basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully estab- lished, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based mo- lecular imaging, it can help to better investigate the invasive behaviors of HCC cells.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

Objective Newly identified human rhinovirus C （HRV-C） and human bocavirus （HBoV） cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.Methods A platform for culturing human airway epithelia in a three-dimensional （3D） pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction {PCR）. Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-2, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial （HAE） cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.Conclusion Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

A Novel <i>in Vitro</i>Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of Breast Cancer Cells

In this work, patterned macropores with a diameter larger than 100 μm were introduced to pristine three-dimensional (3D) nanofibrous bacterial cellulose (BC) scaffolds by using the infrared laser micromachining technique in an attempt to create an in vitro model for the culture of breast cancer cells. The morphology, pore structure, and mechanical performance of the obtained patterned macroporous BC (PM-BC) scaffolds were characterized by scanning electron microscopy (SEM), mercury intrusion porosimeter, and mechanical testing. A human breast cancer cell (MDA-MB-231) line was cultured onto the PM-BC scaffolds to investigate the role of macropores in the control of cancer cell behavior. MTT assay, SEM, and hematoxylin and eosin (H&E) staining were employed to determine cell adhesion, growth, proliferation, and infiltration. The PM-BC scaffolds were found to be able to promote cellular adhesion and proliferation on the scaffolds, and further to allow for cell infiltration into the PM-BC scaffolds. The results demonstrated that BC scaffolds with laser-patterned macropores were promising for the in vitro 3D culture of breast cancer cells.

Effect of deforolimus and VEGF on angiogenesis in endometrial stromal cells following three-dimensional culture

The presence of endometrial tissue outside of the uterine cavity is named endometriosis and is the most common gynecologic disorder in women. Determining the inhibitory effect of a Deforolimus on angiogenesis in a three-dimensional (3-D) culture of human endometrial stromal cells (hEnCs) in vitro. The important mechanism in the pathogenesis of endometriosis is angiogenesis, and deforolimus has been shown to have anti-angiogenic activity. This was an in vitro study of human endometrial stromal cells in 3-D culture of fibrin matrix. Endometrial stromal cells isolated and placed in a 3-D fibrin matrix culture system for angiogenesis with VEGF and inhibit angiogenesis by deforolimus. Finally these cells analyzed by CD31 antibodies. After 3 weeks, in cells treated with VEGF, endothelial cell branching was observed and rudimentary capillary-like structures formed. In the presence of 5μM of deforolimus, angiogenesis was reduced. The deforolimus were shown to be effective in inhibiting the mechanisms of angiogenesis.

Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

Crab cell line,especially continuous crab cell line,can provide us a useful tool for studies on the virology,immunology,and molecular biology of crabs.However,no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of mitosis-arrest.In this study,long-term in vitro culture conditions for both two-(2D)and three-dimensions(3D)were successfully developed for the circulating hemocytes of swimming crab Portunus trituberculatus,designated as PTH cells.In 2D culture,a novel crab basic medium in osmolarity of 990–1100 mOsm/kg was optimized for the first time,which is different from Leibovitz's L-15 medium in mainly the components of amino acids,containing double strengths of the contents of free amino acid mixture in the crab serum.Then an optimal crab growth medium was developed by supplementing 5%fetal bovine serum,50-g/L yeast extract powder,20-μg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium,and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH cells.These 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid nitrogen.In 3D culture,using the same crab growth medium,the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60%on Day 103.This work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.

Application of 3D Scanned Big Data of Large-scale Cultural Heritage Objects Based on Noise-robust Transparent Visualization

Three-dimensional(3D) scanning technology has undergone remarkable developments in recent years.Data acquired by 3D scanning have the form of 3D point clouds.The 3D scanned point clouds have data sizes that can be considered big data.They also contain measurement noise inherent in measurement data.These properties of 3D scanned point clouds make many traditional CG/visualization techniques difficult.This paper reviewed our recent achievements in developing varieties of high-quality visualizations suitable for the visual analysis of 3D scanned point clouds.We demonstrated the effectiveness of the method by applying the visualizations to various cultural heritage objects.The main visualization targets used in this paper are the floats in the Gion Festival in Kyoto(the float parade is on the UNESCO Intangible Cultural Heritage List) and Borobudur Temple in Indonesia(a UNESCO World Heritage Site).

A Three-Dimensional (3D) Environment to Maintain the Integrity of Mouse Testicular Can Cause the Occurrence of Meiosis

Adhesions between different cells and extracellular matrix have been studied extensively in vitro, but little is known about their functions in testicular tissue counterparts. Spermatogonia and their companion somatic cells maintain a close association throughout spermatogenesis and this association is necessary for normal spermatogenesis. In order to keep the relative integrity of the testicular tissues, and to detect the development in vitro, culture testicular tissues in a three- dimensional （3D） agarose matrix was examined. Testicular tissues isolated from 6.5 d postpartum （dpp） mouse were cultured on the top of the matrix for 26 d with a medium height up to 4/5 of the 3D agarose matrix. The results showed that in this 3D culture environment, each type of testicular cells kept the same structure, localization and function as in vivo and might be more biologically relevant to living organisms. After culture, germ cell marker VASA and meiosis markers DAZL and SCP3 showed typical positive analysed by immunofluorescence staining and RT-PCR. It demonstrated that this 3D culture system was able to maintain the number of germ cells and promote the meiosis initiation of male germ cells.

Culture and purification of human fetal olfactory ensheathing cells using different attachment rates combined with intermittent NT3 nutrition

Objective：To explore a simple and pragmatic method to obtain sufficient olfactory ensheathing cells from human fetus by selective attachment of harvested cells combined with intermittent NT3 nutrition. Methods：DMEM/F12 culture solution including 10% fetal bovine serum or NT3 was used to culture olfactory ensheathing cells intermittently every 48 h. The cell state and growth rates of OECs were observed, and P75 staining was used to estimate the purity of the cells. Results：Human fetal OECs were positive with P75 immunocytochemical staining. OECs in dipolar or tripolar shape formed networks by their processes in vitro. The purity of OECs in ＂good state＂ was about 95% at 9 d and 83% on 12 d, respectively. Conclusion：The method of using different attachment rates combined with intermittent NT3 addition is a simple and effective way to culture and purify OECs.

Human Endometrial Stem Cells May Differentiate into Schwann Cells in Fibrin Gel as 3D Culture

Damage in central nervous system plays an important role in biological life and causes severe paralysis of limbs and some organs. There are solutions to problems that can be a great revolution in the transplanted spinal cord and nerve injuries. Schwann cells (SCs) have important roles in development, myelination and regeneration in the peripheral nervous system. The applications of SCs in regenerative medicine are limited because of slow growth rate and difficulties in harvesting. Critical to the hypothesis is the experimental fact that human endometrial-derived stem cells (hEnSCs) as multipotent accessible source of cells are known as useful cell candidates in the field of nerve tissue engineering. We decided to use the three-dimensional culture of Schwann cells differentiated from endometrial stem cell in fibrin gel. In this study, we investigate the expression of differentiated Schwann cell markers by exposing of endometrial stem cells with induction media including FGF2/FSK/HRG/RA. Using immunocytochemistry, we show that differentiated cells express S100 and P75 markers. These results show that for the first time, human endometrial stem cells can be differentiated into Schwann cells in 2D and 3D culture. These novel differentiated cells in fibrin gel might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches for nerve repair.

ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase(ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbedwith a structure resembling the loose connective tissue morphology in a novel 3 D culture system. We confirmed that the 3 D system using CellbedTMaccurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3 D-cultured tongue cancer cell lines than in 2 D cultures. Typically, under conventional 2 D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells.However, in the 3 D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3 D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3 D culture systems using Cellbed? are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

3D bioprinting for cell culture and tissue fabrication

Three-dimensional （3D） bioprinting is a computer-assisted technology which precisely controls spatial position of biomaterials, growth factors and living cells, offering unprecedented possibility to bridge the gap between structurally mimic tissue constructs and functional tissues or organoids. We briefly focus on diverse bioinks used in the recent progresses of biofabrication and 3D bioprinting of various tissue architectures including blood vessel, bone, cartilage, skin, heart, liver and nerve systems. This paper provides readers a guideline with the conjunction between bioinks and the targeted tissue or organ types in structuration and final functionalization of these tissue analogues. The challenges and perspectives in 3D bioprinting field are also illustrated.

微生物合成4-羟基-2,5-二甲基-3(2H)-呋喃酮的研究进展

4-羟基-2,5-二甲基-3(2H)-呋喃酮(HDMF)是一种具有焦糖味的安全优良的甜味香料和增香剂,其阈值低、香势好且特征性强,被广泛应用于食品、烟草等产品中,市场需求量大,尤其是天然来源的HDMF受到人们更多的青睐。然而,天然来源的HDMF产量较低,尚不能满足当前市场的需求。该文从其应用、检测、生产方法、合成HDMF微生物的种类、微生物合成HDMF的机制与关键酶及其发酵培养影响因素进行简述,重点侧重于已报道的具有合成HDMF的微生物种类、微生物合成HDMF的内在机制及其影响其发酵的培养因素,旨在为后续筛选、改造获得优良高产HDMF微生物菌株,并优化微生物发酵条件实现生物法高效制备天然HDMF提供参考。

Effect of chitin-architected spatiotemporal three-dimensional culture microenvironments on human umbilical cord-derived mesenchymal stem cells

Mesenchymal stem cell(MSC)transplantation has been explored for the clinical treatment of various diseases.However,the current two-dimensional(2D)culture method lacks a natural spatial microenvironment in vitro.This limitation restricts the stable establishment and adaptive maintenance of MSC stemness.Using natural polymers with biocompatibility for constructing stereoscopic MSC microenvironments may have significant application potential.This study used chitin-based nanoscaffolds to establish a novel MSC three-dimensional(3D)culture.We compared 2D and 3D cultured human umbilical cord-derived MSCs(UCMSCs),including dif-ferentiation assays,cell markers,proliferation,and angiogenesis.When UCMSCs are in 3D culture,they can differentiate into bone,cartilage,and fat.In 3D culture condition,cell proliferation is enhanced,accompanied by an elevation in the secretion of paracrine factors,including vascular endothelial growth factor(VEGF),hepa-tocyte growth factor(HGF),Interleukin-6(IL-6),and Interleukin-8(IL-8)by UCMSCs.Additionally,a 3D culture environment promotes angiogenesis and duct formation with HUVECs(Human Umbilical Vein Endothelial Cells),showing greater luminal area,total length,and branching points of tubule formation than a 2D culture.MSCs cultured in a 3D environment exhibit enhanced undifferentiated,as well as higher cell activity,making them a promising candidate for regenerative medicine and therapeutic applications.

Rapid fabrication of modular 3D paper-basedmicrofluidic chips using projection-based 3D printing

Paper-based microchips have different advantages,such as better biocompatibility,simple production,and easy handling,making them promising candidates for clinical diagnosis and other fields.This study describes amethod developed to fabricate modular three-dimensional(3D)paper-based microfluidic chips based on projection-based 3D printing(PBP)technology.A series of two-dimensional(2D)paper-based microfluidic modules was designed and fabricated.After evaluating the effect of exposure time on the accuracy of the flow channel,the resolution of this channel was experimentally analyzed.Furthermore,several 3D paper-based microfluidic chips were assembled based on the 2D ones using different methods,with good channel connectivity.Scaffold-based 2D and hydrogel-based 3D cell culture systems based on 3D paper-based microfluidic chips were verified to be feasible.Furthermore,by combining extrusion 3D bioprinting technology and the proposed 3D paper-based microfluidic chips,multiorgan microfluidic chips were established by directly printing 3D hydrogel structures on 3D paperbased microfluidic chips,confirming that the prepared modular 3D paper-based microfluidic chip is potentially applicable in various biomedical applications.

A new method of mesh simplification for 3-Dimension terrain using Laplace operator

This paper proposes a new method to simplify mesh in 3D terrain. The 3D terrain is presented by digital elevation model. First, Laplace operator is introduced to calculate sharp degree of mesh point, which indicates the variation trend of the terrain. Through setting a critical value of sharp degree, feature points are selected. Second, critical mesh points are extracted by an recursive process, and constitute the simplified mesh. Third, the algorithm of linear-square interpolation is employed to restore the characteris- tics of the terrain. Last, the terrain is rendered with color and texture. The experimental results demonstrate that this method can compress data by 16% and the error is lower than 10%.

The 3D Cell Culture System in the Study of Tumor-Applications and Prospects

Compared with 2D tumor cell culture, 3D tumor cell culture can better simulate the microenvironment of signal transduction between cells and extracellular matrix. As one of the best cell models in tumor research, it has been widely used in the study of cancer cell morphology, nanotechnology drug delivery system, and anticancer drug screening. The main theme of this paper is to review the previous research of 3D cell culture applying to tumors in vitro and the prospects for the applications of 3D cell culture system.

PROLIFERATION OF ANTI－CD_3 McAb AND IL－2 INDUCED SPLENOCYTES AND ANTITUMOR EFFECT OF THEIR CULTURE SUPERNATANTS

The proliferation of splenocytes from health adults was induced by anti-CD3 McAb and IL-2.The proliferative potential of the splenocytes and antitumor activity of their culture supernatants of splenocytes were studied.The results showed that anti-CD3 McAb not only enhanced the proliferation of the splenocytes directly,but also enhanceil that of induced by IL-2.Their enhancing effect was more significant when the incubation time in vitro was prolonged.The culture supernatants of anti-CD3 and IL-2 induced splenocytes also had the antitumor activity and enhancing capability to the antitumor activity of LAK tells.The results suggested that LAK cells could secret lymphokine,and this effect would be synergically promoted when anti-CD3 and IL-2 were simultaneously used.

传统文化与创新的融合——基于“1+3+4”插花模式的多元化美育实践探究

传统文化在中华民族历史中扮演着重要角色,它是中华民族的瑰宝,凝结了民族的智慧和文化。当代社会,由于现代化冲击和时尚的影响,许多传统文化元素逐渐淡出人们的生活。在这个背景下,如何将传统文化融入现代生活,使之与创新相互交融,成为一个亟待解决的问题。“1+3+4”插花模式是一种将传统文化与创新相结合的实践模式,打破了传统插花模式的束缚,创造了更加多元化的艺术表现形式。本文以“1+3+4”插花模式为基础,探讨数字化时代多元化美育的实践方式。

Investigation of VEGF and PDGF signals in vascular formation by 3D culture models using mouse ES cells

Vascular formation in vivo involves several processes and signal cascades subsequently occurring in the embryo. Several models by ES cells have been reported for analysis in vitro. We show here a 3D culture system using collagen gel (AteloCell) as a simple and useful system for investigating vascular formations and analyzing the roles of factors in vivo. Although VEGF and PDGF are growth factors with multi-potentials for vascular formation, their sequential roles have not been elucidated. We investigated the effects of VEGF and PDGF B signals for vascular formation by a 3D culture system that embedded embryoid bodies (EBs) from ES cells into a collagen gel. After embedding EBs in the collagen gel with a medium containing VEGF, EBs gave off CD105 immunopositive vessels as the initial step of vasculogenesis. When the factor in the culture medium for EBs was switched from VEGF to PDGF B after 5 days of culture, the morphological features of vessels varied, suggesting the occurrence of vascular-type differentiation. After 11 days of 3D culture, vessels in both groups cultured with VEGF alone and switching to VEGF B at day 5 showed Flk-1 immunoreactivity. Some blood vessels cultured with PDGF B after day 5 expressed either EphrinB2 (arteriole marker) or Flt-4 (lymphatic marker) immunoreactivity, but vessels cultured with VEGF alone exhibited neither of them. Vessels cultured with these two factors could not differentiate into a venous type. The present study indicates that VEGF is the initial signal for vasculogenesis, and that PDGF B is probably involved in vascular diversification.