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Cystic 3D cultures. Closer to living cellular structures



New methods of cell culture increasing the similarity to physiologic tissue conditions are necessary to improve drug investigations and other cell biology investigations, by improving the predictability of biological and metabolic effects to xenobiotics and natural products.

A simplified model of cellular interactions, the 2D co-culture, is obtained just by adding two cell types into the same culture device in which cells, attached to a substrate, contact each other through their borders and extensions struggling for a limited territory. With this model cellular interactions are observed with detail as long as one cell type does not dominate the space and displaces the other one. For example, in structural studies of endothelial cells and mural cells interactions, these co-cultures became extremely demonstrative tools because they show the special interactive space redistribution for both cell types, based on physiologic relations (1)(2)

Co-cultures forming microspheres allow for real cell to cell functional contact, by reaching wider interaction surfaces between cells. In addition studies based on these methods show important results, for example analyzing the establishment of hepatocyte interaction with endothelial cells and other liver connective cells (3). Moreover, the internal cells of the microspheres do not suffer the excessive oxidative environment of the 2D cell cultures exposed to regular 20% atmospheric oxygen concentration (relative hypoxia). Microsphere inner cells have quite a physiologic oxygen tension environment (4).  However, these co-cultures are limited in cell number, final volume and architecture, because the center of the microsphere environment becomes nutrient deficient due to the cell growth and sphere expansion. 

Microfluidic perfusion devices allow for more extensive growth and more accurate inter-cellular interactions (5). Under these tissue-like conditions the formation of quasi realistic cell-cell interactions and cellular physiologic polarization is possible. The only limitations of these cultures are the on edge sampling, limited harvesting and analysis of intra-cellular molecular components (6). Moreover, cell analysis and sorting from microfluidic cultures are severely limited if the culture chip and the specific flow cytometry chip are not integrated (7) (See blog http://celartia.com/cell-culture-blog/advances-in-cell-sorting-taking-care-of-fragile-cells-neuronal-stem-cells-embryonic-cells-and-3d-cultures).

Interestingly, cell cultures developed in rotational microgravity (8) produce substantial population of cells aggregated in 3D structures on carriers (9) or without carriers. Inside these bioreactors, due to the pseudo-microgravity, cells are maintained in suspension under very low-shear stress throughout culture duration. In these devices growing cells form spheroid aggregates expand by increasing the spheroid size and by fusion of multiple spheroids. The architecture of these spheroids varies by cell type. In these bioreactors used, for example with liver cell cultures, cellular interactions induce cell shape changes, mimicking the in-vivo cell shape, forming tissue-like architectures, and increasing the expression of cell-cell adhesion molecules, such as CD44 and E cadherin (8). These bioreactors are functional, easy to assemble and trouble-free to maintain (10)
Cellular interactions, based in released soluble elements, including extracellular organelles, are major components of the cell-cell functional integration without cell-cell contact. Most of these integrations are investigated generally in three steps:
1) Isolation and purification of the target elements released by the cell of interest (cell A).
2) Introduction of this element in the media where cells B are living.
3) Analysis of how the B cells respond to the presence of the soluble element.

Lately, intercellular interactions driven by extracellular organelles such as exosomes, shedding microvesicles (SMVs) and apoptotic blebs (ABs), released into the microenvironment, have become an important research subject (11). Membranous vesicles shed from live cells were first observed in the early 1980s and it was proposed to be just a mechanism through which cells discard inert debris (12).

Exosomes secretion is observed from most cell types, under both physiological and pathological conditions, especially tumor cells and hematopoietic cells, platelets, mast cells, macrophages, dendritic cells, B and T lymphocytes, epithelial cells, fibroblasts, astrocytes, and neurons (13). Exosomes contain cytosolic and membrane proteins derived from the producer cells including targeting/adhesion molecules, membrane trafficking molecules, cytoskeleton molecules, chaperones, signal transduction proteins, cytoplasmic enzymes and many others. Tumor-derived exosomes contain tumor antigens, certain immunosuppressive molecules such as FasL, TRAIL, TGF-β and functional RNA molecules including siRNA (14) all of them potentially involved in physiological signals and functions by intercellular transfer (15).

The exosomes physiology involves the existence of a “sender cell” and a “receiver cell”, both interacting and sharing or alternating the rolls in a real cross talk between them. Therefore, exosomes formation and release from cell X, acting as “sender”, can mediate responses to cell Z acting as “receiver”, that may include changes of the composition and amount of exosomes elaborated and released by this cell Z. Cell Z, now acting as “sender”, may induce additional changes on cell X, now acting as “receiver”. This reciprocal stochastic chain of actions and counteractions play the axial role in animal and organ physiology. Importantly, that kind of feed-back may influence cancer and stromal cell responses to therapeutic agents in a manner not possible to disclose in classic cell culture systems, even if these are used with mixed cell co-cultures.

Exosomes, specifically characterized by having a size of 30 to 100 nm in diameter and a density slightly over water (f 1.13–1.19 g/mL), sediment spontaneously and can be experimentally pelleted at 100,000×g. Aiming to study this sort of intercellular interaction driven through exosomes and other cellular fragments, I decided to develop a system suitable to investigate them, using the simple and unique bioreactor Petaka G3. Conceptually this bioreactor allows: 
1)      Growth of more than one cell type, separated by an inter-space full of media.

2)      Growth of several million cells, letting repeated cell sampling operations without interruption of the culture.

3)      Separate sampling of the different cell types included in the co-culture.

4)     Repeated media renewal and sampling operations without the interruption of the culture.

The biological product of this concept is the “Cystic 3D cell culture” that offers, in a single volume, at least three separate sheets of cells, with or without matrixes, growing with well defined borders, surrounding a pocket (the cyst) with 15 to 25 mL of shared media (see upper figure). The system is a cube like structure with 6 sides or 3 pairs of opposed walls facing each other. In this system the central cavity or cyst receives everything secreted or released by the surrounding cells including metabolites, extracellular organelles, exosomes, shedding microvesicles, apoptotic blebs and others. Cell shits are attached to the walls, uncoated or coated with specific matrix of single molecules such as Laminin, Vitronectin, Collagen, etc. or molecular mixtures such as Matrigel or others, all favoring cell attachment through integrins (16). One side of the device may be coated with a thick layer of semisolid gel allowing the formation of cell clusters (17) and cell penetration evaluation (invasion assays) (18).

Soluble secretions and particulate elements released by cells forming the walls circulate randomly through the cyst media, like in the blastocysts, ovary follicles and others. Those suspended elements become in contact with the other wall cells dragged by soft and slow convection streams, generated by controlled temperature gradients,.  Many physiologic reactions are controlled by soluble molecules and particulate elements, such as exosomes released from living cells (19, 20, 21 and 22)

The bottom side layer of cells is preferably influenced by particulate elements, it receives by gravity sedimentation all those elements having density over 1, released by the upper cells; this influence is even more intensive on all cells living on the side of the cyst when exposed to a multiplied g force vector, if the device is subject to a convenient centrifugation.

With an accurate control of hypoxia, close to physiologic DO (about 2 ppm), CO2 control and dehydration control (Petaka G3 conditions), these cultures could evolve for more than 2 weeks, allowing long term inter-cellular interactions.

The cyst culture system (Petaka G3 device) allows for free-enzyme cell release of all the cells or samples of each cell type separately, therefore sampling each side independently for microscopy, FACS, PCR or biochemistry analysis.

CYSTIC 3D CULTURES-s3sMoreover, magneto-transfection  of cells attached on either side of the cystic system (23), using for gene delivery silica-iron-oxide magnetic nanoparticles (24) and a confined magnetic field, allows separate transfection of each monolayer (see side figure.). Furthermore, that transfection can be limited to part of the monolayer, it can be repeated several times on the same monolayer or it can be done in different segments of it in a targeted manner (25).  

After magneto-transfection cells can be partially released without enzymes, magneto-selected and concentrated for expansion, further analysis or treatment (26)

1.            Evensen L, Micklem DR, Blois A, Berge SV, Aarsæther N, Littlewood-Evans A, et al. Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation. PLoS ONE. 2009 Jun 4;4(6):e5798.

2.            Talasila KM, Brekka N, Mangseth K, Stieber D, Evensen L, Rosland GV, et al. Tumor versus Stromal Cells in Culture—Survival of the Fittest? PLoS ONE. 2013 Dec 2;8(12):e81183.

3.            Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013;87:1315–530.

4.            Lambrechts D, Roeffaers M, Kerckhofs G, Roberts SJ, Hofkens J, Van de Putte T, et al. Fluorescent oxygen sensitive microbead incorporation for measuring oxygen tension in cell aggregates. Biomaterials. 2013 Jan;34(4):922–9.

5.            Böttger J, Schütte J, Benz K, Freudigmann C, Hagmeyer B, Höppner C, et al. A Microfluidic 3D Hepatocyte Culture System -HepaChip®- For Drug And Toxicity Investigations. Z Für Gastroenterol [Internet]. 2012 Jan 9 [cited 2013 Dec 21];50(01). Available from: https://www.thieme-connect.com/ejournals/abstract/10.1055/s-0031-1295800

6.            Kim J, Johnson M, Hill P, Gale BK. Microfluidic sample preparation: cell lysis and nucleic acid purification. Integr Biol. 2009 Sep 30;1(10):574–86.

7.            Mao X, Nawaz AA, Lin S-CS, Lapsley MI, Zhao Y, McCoy JP, et al. An integrated, multiparametric flow cytometry chip using “microfluidic drifting” based three-dimensional hydrodynamic focusing. Biomicrofluidics. 2012 Apr 20;6(2):024113–024113–9.

8.            Ingram M, Techy GB, Saroufeem R, Yazan O, Narayan KS, Goodwin TJ, et al. Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor. In Vitro Cell Dev Biol Anim. 1997 Jun;33(6):459–66.

9.            Lei X, Ning L, Cao Y, Liu S, Zhang S, Qiu Z, et al. NASA-Approved Rotary Bioreactor Enhances Proliferation of Human Epidermal Stem Cells and Supports Formation of 3D Epidermis-Like Structure. PLoS ONE [Internet]. 2011 Nov 9 [cited 2013 Dec 21];6(11). Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3212516/

10.          GEN | Magazine Articles: Rotating Bioreactors for Manufacturing [Internet]. GEN. [cited 2013 Dec 22]. Available from: http://www.genengnews.com/gen-articles/rotating-bioreactors-for-manufacturing/1899/

11.          Mathivanan S, Ji H, Simpson RJ. Exosomes: Extracellular organelles important in intercellular communication. J Proteomics. 2010 Sep 10;73(10):1907–20.

12.          Pan BT, Teng K, Wu C, Adam M, Johnstone RM. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol. 1985 Sep 1;101(3):942–8.

13.          Yang C, Robbins PD. The Roles of Tumor-Derived Exosomes in Cancer Pathogenesis. Clin Dev Immunol [Internet]. 2011 Nov 30 [cited 2013 Dec 22];2011. Available from: http://www.hindawi.com/journals/cdi/2011/842849/abs/

14.          Shtam TA, Kovalev RA, Varfolomeeva EY, Makarov EM, Kil YV, Filatov MV. Exosomes are natural carriers of exogenous siRNA to human cells in vitro. Cell Commun Signal CCS. 2013;11:88.

15.          Marjon KD, Gillette JM. Measurement of intercellular transfer to signaling endosomes. Methods Enzymol. 2014;534:207–21.

16.          Lim JJ, Lee DR, Song H-S, Kim K-S, Yoon TK, Gye MC, et al. Heparin-binding epidermal growth factor (HB-EGF) may improve embryonic development and implantation by increasing vitronectin receptor (integrin alphanubeta3) expression in peri-implantation mouse embryos. J Assist Reprod Genet. 2006 Mar;23(3):111–9.

17.          Mathupala SP, Sloan AE. An agarose-based cloning-ring anchoring method for isolation of viable cell clones. BioTechniques. 2009 Apr;46(4):305–7.

18.          Hall DMS, Brooks SA. In vitro invasion assay using matrigelTM: a reconstituted basement membrane preparation. Methods Mol Biol Clifton NJ. 2014;1070:1–11.

19.          Bianco NR, Kim SH, Ruffner MA, Robbins PD. Therapeutic effect of exosomes from indoleamine 2,3-dioxygenase–positive dendritic cells in collagen-induced arthritis and delayed-type hypersensitivity disease models. Arthritis Rheum. 2009;60(2):380–9.

20.          Gansuvd B, Hagihara M, Higuchi A, Ueda Y, Tazume K, Tsuchiya T, et al. Umbilical cord blood dendritic cells are a rich source of soluble HLA-DR: synergistic effect of exosomes and dendritic cells on autologous or allogeneic T-Cell proliferation. Hum Immunol. 2003 Apr;64(4):427–39.

21.          Bu N, Li Q-L, Feng Q, Sun B-Z. Immune protection effect of exosomes against attack of L1210 tumor cells. Leuk Lymphoma. 2006 Jan;47(5):913–8.

22.          Clayton A, Mitchell JP, Court J, Mason MD, Tabi Z. Human Tumor-Derived Exosomes Selectively Impair Lymphocyte Responses to Interleukin-2. Cancer Res. 2007 Aug 1;67(15):7458–66.

23.          Plank C, Anton M, Rudolph C, Rosenecker J, Krötz F. Enhancing and targeting nucleic acid delivery by magnetic force. Expert Opin Biol Ther. 2003 Aug;3(5):745–58.

24.          Mykhaylyk O, Sobisch T, Almst&auml, tter I, Sanchez-Antequera Y, Brandt S, et al. Silica-Iron Oxide Magnetic Nanoparticles Modified for Gene Delivery: A Search for Optimum and Quantitative Criteria. Pharm Res. 2012;29(5):1344 – 1365.

25.          Hofmann A, Wenzel D, Becher UM, Freitag DF, Klein AM, Eberbeck D, et al. Combined targeting of lentiviral vectors and positioning of transduced cells by magnetic nanoparticles. Proc Natl Acad Sci. 2009 Jan 6;106(1):44–9.

26.          Sanchez-Antequera Y, Mykhaylyk O, Til NP van, Cengizeroglu A, Jong JH de, Huston MW, et al. Magselectofection: an integrated method of nanomagnetic separation and genetic modification of target cells. Blood. 2011 Apr 21;117(16):e171–e181. 
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