Brochure NanoCulture® Plates

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Matrix-free and adhesion-type 3D cell culture Micro-structure as a scaffold for cells

100µm

Spheroid size of HepG2 cells over time. Box plot of spheroid diameter from HepG2 cells on a 96-well NCP, seeded at 10,000 cells per well. A linear spheroid growth is observed over a one week time period, after which the size reaches a plateau.

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Cells seeded on NCP/NCD sediment to the well bottom and attach to the pattern. Cells are allowed to migrate randomly and dynamically because of the controlled limited adhesion strength to the patterned film. When cells migrate together, cell-cell interactions prevail over cell-scaffold interactions, which in turn results in spheroid formation.

Schematic diagram of spheroid formation on NCP/NCD

Visual changes of spheroid formation by HT29 cells over time

Spheroid formation mechanism on NanoCulture Plates / Dishes (NCP / NCD)

attachment to the bottom

migration cell-cell interaction

Electron microscopy picture of a spheroid (HeLa cells) attaching to the nano-scale pattern on the film. The pattern provides a scaffold for the cells to grab hold of by pseudopodia. At the same time the physical and chemical nature of the pattern inhibits unwanted 2-dimensional spreading of the cells. Pictures courtesy of Prof. Hideo Namiki, Waseda University.

Well-bottom film with nano-scale pattern. The rainbow colors are caused by the light interference of the nano-scale pattern.

NanoCulture Plates/Dishes (NCP/NCD) are engineered with a special plastic film bottom, on which nano-scale structures are evenly patterned on the well bottom surface. The pattern mimics the extracellular matrix. The chemical and physical properties of the nano-scale structures allow controlling of the adhesion of the cells to the film and provide a scaffold for the cells.

1culture days

User-friendly 3D cell culture compatible with various assays

Various well-sizes of NanoCulture series

NCP (24, 96, 384 well) and NCD (35 mmΦ)

Advantages of NanoCulture Plates/Dishes

NanoCulture Plate / Dish are available in 24, 96 and 384-well plates and in 35 mm dishes to fit various applications.

protein/ RNA extraction

cell-based assay/ HTSapplication example

well bottom area (mm2)

35 mm dish 24 well plate 96 well plate 384 well plate754 193 34 10

Uniformity

Microscopic imaging

Cell Viability

Free of coating reagents and biological materials. Made of fully synthetic polymeric material, with very high lot-to-lot reproducibility.

Lack of highly viscous or rigid 3D environment allows high cell mobility and rapid spheroid formation. Spheroids can be observed 3 day post-seeding already.

Rapid Spheroid Formation

Automation

Ready-to-Use Same as conventional monolayer culture plates. Just add cell suspension into the wells for seeding. Simple, convenient and easy to automate.

Large scale spheroid generation can be achieved by automated cell culture using the standard plate formats compatible with robots conventionally used for traditional monolayer culture.

Cell-cell interactions create a natural microenvironment for the cells, resulting in high cell viability.

Morphologies of spheroids cultured on NanoCulture Plate/Dish

Cells form 3D structures with various morphologies, depending on cell specific characteristics (migration, proliferation, intercellular adhesion molecule expression, degree of differentiation). Typically, the structures can be classified into 4 types.

spheroids with smooth surface

spheroids with rough and bumpy surface

grapevine-like hollow formation

Capan-2 PC-3 SK-BR-3 MCF-7Data provided by: Prof. Hideo Namiki, Waseda University

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Inherent parfocal localization of all spheroids at the well bottom. Optical transparency of the film allows direct and automatable fluorescence/luminescence imaging.

In vitro tumor modelingNatural formation of hypoxic regions

Sensitivity difference against Trastuzumab between 2D and 3DHuman breast cancer cell line BT474 showed stronger sensitivity against Anti-HER2 antibody Trastuzumab (10μg/mI). Viability was measured by ATP assay, DNA quantitation, and cell counting.

Drug sensitivity studies show that responses to anticancer drugs differ substantially between 2D monolayer culture versus 3D NCP culture. Spheroids grown on NCP are morphologically and metabolically similar to in vivo tumors, thus offering an advanced model for drug sensitivity assays and drug response evaluation in primary tumors.

Different anticancer drug sensitivity compared to monolayer culture

Hypoxia region inside spheroidStable cell line expressing GFP regulated by HIF enhancer (HRE) was cultured for 7 days on NCP. Activation of HIF can be observed inside the spheroids.

In vivo, tumors exhibit hypoxic regions caused by rapid proliferation of cells and scarce blood supply. These hypoxic regions are known to be more resistant against radio- and chemotherapy.

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Primary tumor cells form spheroids

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Cancer cells grow alongside interstitial cells in vivo, but co-culturing primary tumor cells with fibroblasts often results in an overgrowth by the latter. On NCP, co-culture of primary tumor cells with associated fibroblasts has been demonstrated, without problems of overgrowth. Over 100 types of primary tumor cells have been cultured on NCP in collaboration with the Japanese National Cancer Research Center.

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) Drug sensitivity difference between 2D and 3DApproximately 1,300 compounds were tested on human A549 lung cancer cell line both in 2D and 3D conditions. Lowered drug sensitivity in 3D system compared to 2D is indicated in blue, higher in red, and no significant difference in gray.

Data provided by Dr. Yukie Yoshii of National Institute of Radiological Sciences

Spheroids cultured on NCP also exhibit naturally formed hypoxic regions, and therefore display similar metabolic pathways as in vivo tumor cells do. This makes spheroids a much more physiologically relevant in vitro tumor model than conventional monolayer cell culture.

Culture of primary human pancreatic cancer cells with interstitial cells. Spheroid formation is observed only on NCP (right), but not on 2D plate (left). The proliferation of interstitial cells seemed to be controlled on NCP. Data provided by Dr. Tetsuya Nakatsura of the Japanese National Cancer Center.

Compounds provided by The University of Tokyo Open Innovation Center for Drug Discovery

Normal cells form spheroids on NCP

Differentiation of human mesenchymal stem cells UET-13 into adipocytesHuman immortalized mesenchymal stem cells UET-13 cultured in both 2D and 3D culture condition, with induced differentiation into adipocytes. By staining lipid-droplets with Oil red O, most of the cells were stained red in case of 3D culture. Also, marker gene expressions showed quicker and stronger increase, compared to 2D culture, which indicates higher efficiency of differentiation into mature adipocytes under 3D culture condition.

Promote induction of differentiation efficiency with mesenchymal stem

In addition to cancer cells, normal cells also form spheroids on NCP. Intercellular networks and surface adhesion increase with spheroid formation, resulting in increased cell differentiation potency and differential expression of metabolic enzymes and cytokines. Unlike cancer cells, regular cells grown on NCP stop expanding following spheroid formation.

Cardiac muscle cells spheroid start pulsation

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Physiological activity of liver cells

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secretion of albmin mRNA expression of CYP genes

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Spheroid of primary rat hepatocyte Hepatocytes obtained from rat form spheroids on NCP.

Enhancement of immortalized human hepatocytes functionHigher level of albumin secretion or gene expression of CYP iso-enzymes, compared to 2D culture condition, under 3D culture condition.

Fibroblasts form spheroids

100µm 100µm

Primary rat cardiac myocytes form spheroidsRat cardiac myocytes form spheroids on NCP. Pulsation was also observed.

Fibroblasts form spheroidsHigh motility of fibroblast on NCP surface to form spheroids. No significant proliferation like cancer for cells was observed.

Data provided by Dr. Hajime Okita of National Research Institute for Child Health and Development

NanoCulture product related reagent

Hypoxia Probe is a red phosphorescent substance, which signal is attenuated by oxygen quenching in atmospheric (20% O₂) condition. Under low-oxygen atmosphere, it starts emitting red light. Therefore, this probe can be used to visualize hypoxic conditions within the spheroids, in a homogeneous assay, just byadding it to media.

Hypoxia Probe　

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Oxygen quenching of Hypoxia ProbeUnder atmospheric condition, red phosphorescent signal of Hypoxia Probe is quenched (O₂ and aerated), while emitting red light by argon gas (Ar) substitution.

Hypoxic condition inside spheroidsIt was confirmed that the inside of highly cohesive spheroids has strong hypoxic signal (BT474), while low cohesive cell aggregates do not become hypoxic (PANC-1 and MIA PaCa-2).

BT474 PANC-1 MIAPaCa-2

Data provided by Prof. Seiji Tobita, Gumma University

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There are two patterns, Microsquare and Micorohoneycomb. Which one should I use?

We have evaluated more that 60 cell lines, and confirmed pattern “preference” with approximately 10% of the cell types. (These results are exhibited in our website. Please visit http://www.scivax.com)For more information, please inquire about both patterns of NCP/NCD with your cell types.

Microsquare pattern Microhoneycomb pattern

Structure of culture surfaceLeft： Mirosquare (MS)Right： Microhoneycomb (MH)

Example of superior spheroid formation using microsquare pattern

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Which adhesion type NCP/NCD should I use? Low-binding or high-binding?

First time users of NCP/NCD are recommended to use low-binding type plates. Many of the cell lines adhere strongly to the well-bottom film in case of high-binding plates, resulting in monolayer growth. However, high binding plates may improve cell adhesion of cell types with very week spheroid adhesion to the culture surface.

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FAQ.

Low-binding type High-binding type

Cell A

Cell B

High-binding type is suitable for cell A

Low-binding type is suitable for cell B

What is the difference between 3D cell culture using NCP/NCD and other 3D cultures?Q3.

+ - - + ++ - - +/-+ - + ++ + - +/-

- + +

Easy Handling

Imaging convenience

Lot-to-lot consistency

High Cell Viability

Fast Spheroid Formation

Wide variety of Well Sizes + +

+/- +

+/- + - +/- -

NCP/NCDCompetitive Products

ECM embedding Hanging drop Ultra Low Adhesion U-bottom

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PRODUCT DESCRIPTION FORMAT REFERENCE PACKAGE SIZE

NanoCulture® Plate MS Pattern, Low-Binding

24 well plateNCP-LS-24-2 2 plates

NCP-LS-24-10 10 plates





NanoCulture® Plate MH Pattern, Low-Binding

24 well plateNCP-LH-24-2 2 plates

NCP-LH-24-10 10 plates





NanoCulture® Plate MS Pattern, High-Binding

24 well plateNCP-HS-24-2 2 plates

NCP-HS-24-10 10 plates





NanoCulture® Plate MH Pattern, High-Binding

24 well plateNCP-HH-24-2 2 plates

NCP-HH-24-10 10 plates





NanoCulture® Plate MH&MS Pattern, Low-Binding24 well plate NCP-LSH-24-2 2 plates (1 each)

96 well plate NCP-LSH-96-2 2 plates (1 each)

NanoCulture® Dish MS Pattern, Low-Binding 35 mm dish NCD-LS35-5 5 dishes

NanoCulture® Dish MH Pattern, Low-Binding 35 mm dish NCD-LH35-5 5 dishes

Spheroid Lysis Buffer bottle SLB 2x 7.5 ml

Hypoxia Probe vial LOX-1 2 mg

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JSR Micro N.V. Technologielaan 8 B-3001 Leuven Belgium www.jsrlifesciences.com [email protected]

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Brochure NanoCulture® Plates