Standardní kultivace nádorových buněčných linií ve 2D uspořádání je dobře zavedeným a finančně dostupným experimentálním mode-lem pro in vitro testování biologických účinků potenciálních protinádorových léčiv. 2D kultury však postrádají metabolické a proliferační gradienty, důležité buněčné interakce a signalizace, které jsou přítomné in vivo. 3D buněčné sféroidy zohledňují gradienty živin, kyslíku či odpadních metabolitů, důležitost interakcí mezi buňkami a extracelulární matrix a navozují tak situaci bližší reálným podmínkám. Bio-logické vlastnosti 3D sféroidů a jejich odpovědi na účinky léčiv se značně liší ve srovnání s 2D kulturami. Hodnocením protinádorových účinků potenciálních léčiv na 3D kulturách se zásadně zvyšuje šance na výběr farmakologicky relevantních struktur a snížit tak riziko neúspěchu v průběhu klinického testování.

Traditional cultivation of cancer cell lines in 2D arrangement is well established and affordable experimental model for in vitro testing of biological effects of potential anticancer drugs. However, 2D cultures lack metabolic and proliferative gradients, important cell interac-tions and signaling that are present in vivo. Within 3D spheroids the gradients of nutrients, oxygen or waste metabolites, the importance of interactions between the cells and the extracellular matrix are included, and thus 3D can better simulate in vivo tumor microenviro-ment. The biological properties of 3D spheroids and their responses to drug effects vary greatly compared to 2D cultures. The evaluation of anticancer drug effects on 3D spheroids increases the chances of selection of pharmacologically relevant structures and thus reduce clinical trial failure risk.

• Klíčová slova
• solidní nádory,
• MeSH
• biologické modely MeSH
• buněčné sféroidy * fyziologie   klasifikace   účinky léků   MeSH
• lidé MeSH
• nádorové buňky kultivované cytologie   mikrobiologie   MeSH
• nádory diagnostické zobrazování   MeSH
• preklinické hodnocení léčiv MeSH
• protinádorové látky farmakokinetika   MeSH
• techniky 3D buněčné kultury metody   MeSH
• techniky in vitro metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

In the rapidly evolving landscape of cell biology and biomedical research, three-dimensional (3D) cell culture has contributed not only to the diversification of experimental tools available but also to their improvement toward greater physiological relevance. 3D cell culture has emerged as a revolutionary technique that bridges the long-standing gap between traditional two-dimensional (2D) cell culture and the complex microenvironments found in living organisms. By providing conditions for establishing critical features of in vivo environment, such as cell-cell and cell-extracellular matrix interactions, 3D cell culture enables proper tissue-like architecture and differentiated function of cells. Since the early days of 3D cell culture in the 1970s, the field has witnessed remarkable progress, with groundbreaking discoveries, novel methodologies, and transformative applications. One particular 3D cell culture technique has caught the attention of many scientists and has experienced an unprecedented boom and enthusiastic application in both basic and translational research over the past decade - the organoid technology. This book chapter provides an introduction to the fundamental concepts of 3D cell culture including organoids, an overview of 3D cell culture techniques, and an overview of methodological- and protocol-oriented chapters in the book 3D Cell Culture.

• MeSH
• biomedicínský výzkum * MeSH
• buněčné kultury metody   MeSH
• organoidy * MeSH
• techniky 3D buněčné kultury MeSH
• translační biomedicínský výzkum MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 μm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, β-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.

• MeSH
• buněčná diferenciace * fyziologie   MeSH
• buněčné kultury metody   MeSH
• buněčné linie MeSH
• buněčné sféroidy * metabolismus   MeSH
• myši MeSH
• neurony metabolismus   MeSH
• prionové nemoci * metabolismus   patologie   MeSH
• priony metabolismus   MeSH
• techniky 3D buněčné kultury metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

3D cell culture is an invaluable tool in developmental, cell, and cancer biology. By mimicking crucial features of in vivo environment, including cell-cell and cell-extracellular matrix interactions, 3D cell culture enables proper structural architecture and differentiated function of normal tissues or tumors in vitro. Thereby 3D cell culture realistically models in vivo tissue conditions and processes, and provides in vivo like responses. Since its early days in the 1970s, 3D cell culture has revealed important insights into mechanisms of tissue homeostasis and cancer, and accelerated translational research in cancer biology and tissue engineering.

• MeSH
• biomimetika MeSH
• bioprinting MeSH
• buněčné kultury metody   MeSH
• lidé MeSH
• tkáňové inženýrství MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Východiska: Současné in vitro modelové systémy plně neodrážejí biologickou a klinickou diverzitu karcinomu prostaty (prostate cancer – PCa). Organoidy jsou 3D in vitro buněčné kultury, které mohou lépe rekapitulovat heterogenitu onemocnění a zachovat vlastnosti původního nádoru. Krátkodobá ex vivo kultivace PCa tkání může také usnadnit testování léčiv v personalizované medicíně. Materiál a metody: Pro organoidní kultivaci jsme zpracovali jak nádorovou, tak normální tkáň od 50 pacientů, kteří podstoupili radikální prostatektomii nebo transuretrální resekci prostaty. Kromě toho jsme využili techniku ex vivo tkáňové kultivace a provedli krátkodobý experiment s použitím gemcitabinu a inhibitoru Chk1 MU380 ve vzorcích od 10 pacientů. Výsledky: Celkem jsme byli schopni kultivovat organoidy z 58 % nádorových (29/50) a 69 % normálních tkání (20/29). Imunohistochemické barvení dvou reprezentativních případů odhalilo buněčnou pozitivitu na pan-cytokeratin potvrzující přítomnost epiteliálních buněk. Nadměrná exprese proteinů AMACR a ERG v nádorech však nebyla zachována v organoidech. Dalším omezením bylo udržení organoidů pouze do první pasáže, obvykle po dobu 3 týdnů. Dále byly provedeny krátkodobé testy v ex vivo kultuře nádorových tkání od deseti pacientů. Tkáňové vzorky z prostatektomií většinou vykazovaly nízkou míru proliferace a Ki-67 pozitivity. Další nevýhodou tohoto přístupu byla nekonzistentní morfologie mezi jednotlivými tkáňovými fragmenty. Pouze jeden případ vykazoval vysokou míru proliferace pro testování léčiv a nádorová tkáň byla přítomna ve všech testovaných vzorcích. V naší práci také poskytujeme přehled nedávných studií a podrobné srovnání kultivačních podmínek. Závěr: Podařilo se nám ustavit kultury organoidů i fragmentů tkání z primárních nádorů prostaty. Exprese nádorových markerů však nebyla zachována v získaných organoidech. Nekonzistentní morfologie a nízká proliferace ztěžovaly interpretaci výsledků testování léčiv u většiny případů. Přesto mohou být tyto přístupy slibné při použití tkání z metastatického kastračně rezistentního karcinomu prostaty.

Background: Current in vitro model systems do not fully reflect the biological and clinical diversity of prostate cancer (PCa). Organoids are 3D in vitro cell cultures that may better recapitulate disease heterogeneity and retain parental tumor characteristics. Short-term ex vivo culture of PCa tissues may also facilitate drug testing in personalized medicine. Materials and methods: For organoid culture, we have processed both cancer and normal tissues from 50 patients who underwent radical prostatectomy or transurethral resection of the prostate. In addition, we exploited the ex vivo tissue culture technique and performed short-term chemotherapy assay using gemcitabine and Chk1 inhibitor MU380 in 10 patient samples. Results: In total, we were able to cultivate organoids from 58% of tumors (29/50) and 69% of normal tissue (20/29). Immunohistochemical staining of two representative cases revealed cell positivity for pan-cytokeratin confirming the presence of epithelial cells. However, the overexpression of AMACR and ERG proteins in tumors was not recapitulated in organoids. Another limitation was the propagation of organoids only up to 3 weeks till the first passage. Next, a short-term drug test was performed for ten patients using ex vivo tissue culture. Samples from prostatectomies mostly presented a low proliferation rate as assessed by Ki-67 staining. Another drawback of this approach was inconsistent tissue morphology among particular tissue fragments. Only one case showed a high proliferation rate for drug testing and tumor tissue was present in all tested samples. In our work, we also provide an overview of recent studies and a detailed comparison of culture conditions. Conclusion: We have established cultures of both organoids and tissue fragments from PCa patient samples. However, the expression of tumor markers was not recapitulated in organoids. Inconsistent morphology among tissue fragments and low proliferation hampered the interpretation of the drug testing in most cases. Still, these approaches may be promising using tissues from metastatic castration-resistant prostate cancer.

• Klíčová slova
• ex vivo,
• MeSH
• individualizovaná medicína metody   MeSH
• klinická studie jako téma MeSH
• kultivační techniky MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádory prostaty * patologie   MeSH
• organoidy metabolismus   patologie   MeSH
• transuretrální resekce prostaty MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

Repairing and regenerating damaged tissues or organs, and restoring their functioning has been the ultimate aim of medical innovations. 'Reviving healthcare' blends tissue engineering with alternative techniques such as hydrogels, which have emerged as vital tools in modern medicine. Additive manufacturing (AM) is a practical manufacturing revolution that uses building strategies like molding as a viable solution for precise hydrogel manufacturing. Recent advances in this technology have led to the successful manufacturing of hydrogels with enhanced reproducibility, accuracy, precision, and ease of fabrication. Hydrogels continue to metamorphose as the vital compatible bio-ink matrix for AM. AM hydrogels have paved the way for complex 3D/4D hydrogels that can be loaded with drugs or cells. Bio-mimicking 3D cell cultures designed via hydrogel-based AM is a groundbreaking in-vivo assessment tool in biomedical trials. This brief review focuses on preparations and applications of additively manufactured hydrogels in the biomedical spectrum, such as targeted drug delivery, 3D-cell culture, numerous regenerative strategies, biosensing, bioprinting, and cancer therapies. Prevalent AM techniques like extrusion, inkjet, digital light processing, and stereo-lithography have been explored with their setup and methodology to yield functional hydrogels. The perspectives, limitations, and the possible prospects of AM hydrogels have been critically examined in this study.

• MeSH
• 3D tisk MeSH
• bioprinting metody   MeSH
• buněčné kultury MeSH
• hydrogely * chemie   MeSH
• lékové transportní systémy MeSH
• lidé MeSH
• techniky 3D buněčné kultury metody   MeSH
• tkáňové inženýrství * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

• MeSH
• anatomické modely MeSH
• bioprinting * MeSH
• lidé MeSH
• regenerativní lékařství * MeSH
• techniky 3D buněčné kultury metody   MeSH
• transplantáty MeSH
• umělá inteligence MeSH
• Check Tag
• lidé MeSH

The consideration of human and environmental exposure to dendrimers, including cytotoxicity, acute toxicity, and cell and tissue accumulation, is essential due to their significant potential for various biomedical applications. This study aimed to evaluate the biodistribution and toxicity of a novel methoxyphenyl phosphonium carbosilane dendrimer, a potential mitochondria-targeting vector for cancer therapeutics, in 2D and 3D cancer cell cultures and zebrafish embryos. We assessed its cytotoxicity (via MTT, ATP, and Spheroid growth inhibition assays) and cellular biodistribution. The dendrimer cytotoxicity was higher in cancer cells, likely due to its specific targeting to the mitochondrial compartment. In vivo studies using zebrafish demonstrated dendrimer distribution within the vascular and gastrointestinal systems, indicating a biodistribution profile that may be beneficial for systemic therapeutic delivery strategies. The methoxyphenyl phosphonium carbosilane dendrimer shows promise for applications in cancer cell delivery, but additional studies are required to confirm these findings using alternative labelling methods and more physiologically relevant models. Our results contribute to the growing body of evidence supporting the potential of carbosilane dendrimers as vectors for cancer therapeutics.

• MeSH
• dánio pruhované MeSH
• dendrimery * toxicita   MeSH
• lidé MeSH
• nádory * farmakoterapie   MeSH
• techniky 3D buněčné kultury MeSH
• tkáňová distribuce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Východiska: Primární lidské B buňky chronické lymfocytární leukemie (CLL) podléhají při kultivaci in vitro buněčné smrti, nicméně jejich přežití lze signifikantně prodloužit kontaktem se stromálními buňkami nebo přítomností specifických solubilních faktorů. Pro účely výzkumu chování CLL buněk jsme vytvořili 3D in vitro model, ve kterém bylo simulováno vhodné mikroprostředí pro CLL buňky umožňující studium mechanizmu jejich přežívání v dlouhodobé kultivaci. Materiál a metody: Naším cílem bylo, aby struktura scaffoldu byla geometricky podobná 3D morfologii kostní dřeně, která vyplňuje trabekulární kost, aby měl 3D scaffold dostatečně velký povrch pro zachycení buněk a zároveň velkou pórovitost pro buněčnou migraci a transport živin. Dalším požadavkem byla také alespoň částečná transparentnost potřebná pro pozorování buněčného modelu pomocí optických metod. Připravili jsme 3D scaffoldy z porózního hydrogelu poly (2-hydroxyetyl metakrylát) (pHEMA), poly (2-hydroxyetyl metakrylát-co-2-aminoetyl metakrylát) p (HEMA-co-AEMA) a p (HEMA-co-AEMA) modifikovaný s často používaným adhezním peptidem Arg-Gly-Asp (RGD). Všechny hydrogelové scaffoldy byly vyrobeny ve čtyřech velikostech pórů (125, 200, 300 a 350–450 μm). Scaffoldy byly testovány pomocí HS-5 buněčné linie odvozené z lidských stromálních buněk kostní dřeně a HEK293 buněčné linie odvozené z lidských embryonálních buněk ledvin. Výsledky: Hydrogelový scaffold p (HEMA-co-AEMA) modifikovaný adhezním peptidem Arg-Gly-Asp (RGD) s velikostí pórů 350–450 μm prokázal, že je vhodným systémem pro 3D kultivace buněk, neboť podporuje interakce mezi buňkami navzájem a také mezi buňkami a materiálem. Tento scaffold byl použit pro nasazení kultivace složené z HS-5 buněk a CLL buněk, které byly stimulovány pomocí ligandu CD40 a cytokinu IL-4. Viabilita CLL buněk byla vyšší v přítomnosti obou stimulátorů zároveň než v případě každého zvlášť. Závěr: Ukázali jsme, že technologie 3D scaffoldů je velmi dobře využitelná pro modelování mikrosystémů, kde se nádorové buňky chovají jako ve svém přirozeném mikroprostředí. Klíčová slova: hematoonkologie – leukemie – hydrogel – stromální buňky

Background: Primary human B cells chronic lymphocytic leukemia undergoes apoptosis, from which they can be rescued by contact with stromal cells or by the addition of specific soluble factor, when cultured in vitro. For research purposes of the behavior of CLL cells we created 3D in vitro model in which we simulated appropriate microenvironment for CLL cells to allow study the mechanism of survival of these cells in long-term cultivation. Material and Methods: Our aim was the scaffold structure to be geometrically similar to the 3D morphology of supporting bone marrow tissue in a trabecular bone; the 3D scaffold was also designed to conform to biocompatibility, sufficiently large surface area for cell attachment, high porosity for cell migration, proliferation and transport of nutrients. Another requirement was a partial transparency for inspection of cell model with optical techniques. We prepared 3D scaffolds from porous hydrogel poly (2-hydroxyethyl methacrylate) (pHEMA), poly (2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) p (HEMA-co-AEMA) and p (HEMA-co-AEMA) modified with frequently used cell adhesion peptide Arg-Gly-Asp (RGD). All hydrogel scaffolds were manufactured in four pore diameters (125, 200, 300 and 350–450 μm). Scaffolds were tested with human bone marrow stromal cell line HS-5 and human embryonic kidney cell line HEK293. Results: Hydrogel scaffold p (HEMA-co-AEMA) modified with adhesion peptide Arg-Gly-Asp (RGD) with pore diameter of 350–450 μm demonstrated that it is a convenient system for 3D cell cultivation, since it promotes interaction between the cells and also between the cells and the material. This scaffold was used for seeding of co-cultivation system of HS-5 cells with CLL-cells, which were stimulated through the CD40L signaling pathway as well as via the IL-4 pathway. Viability of B-CLL cells was higher in the presence of both stimulators than with each alone. Conclusions: We have shown that 3D scaffold technology is very useful for modeling of microsystems where the cancer cells behave like in their natural microenvironment. Key words: hematooncology – leukemia – hydrogel – stromal cells This work was supported by grant COST CZ LD15144 “Cellular and acellular grounds for regeneration of bones and teeth” awarded by the Ministry of Education, Youth and Sport of the Czech Republic. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 6. 3. 2017 Accepted: 26. 3. 2017

• MeSH
• biokompatibilní materiály MeSH
• biologické modely MeSH
• chronická lymfatická leukemie patologie   MeSH
• hydrogely * MeSH
• kultivační techniky metody   MeSH
• mezenchymální kmenové buňky MeSH
• nádorové buňky kultivované * MeSH
• nádorové mikroprostředí MeSH
• techniky in vitro MeSH
• tkáňové podpůrné struktury * MeSH
• Publikační typ
• práce podpořená grantem MeSH

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of neoplastic mature B cells in blood, bone marrow, lymph nodes, and spleen. The disease biology remains unresolved in many aspects, including the processes underlying the disease progression and relapses. However, studying CLL in vitro poses a considerable challenge due to its complexity and dependency on the microenvironment. Several approaches are utilized to overcome this issue, such as co-culture of CLL cells with other cell types, supplementing culture media with growth factors, or setting up a three-dimensional (3D) culture. Previous studies have shown that 3D cultures, compared to conventional ones, can lead to enhanced cell survival and altered gene expression. 3D cultures can also give valuable information while testing treatment response in vitro since they mimic the cell spatial organization more accurately than conventional culture. METHODS: In our study, we investigated the behavior of CLL cells in two types of material: (i) solid porous collagen scaffolds and (ii) gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We studied CLL cells' distribution, morphology, and viability in these materials by a transmitted-light and confocal microscopy. We also measured the metabolic activity of cultured cells. Additionally, the expression levels of MYC, VCAM1, MCL1, CXCR4, and CCL4 genes in CLL cells were studied by qPCR to observe whether our novel culture approaches lead to increased adhesion, lower apoptotic rates, or activation of cell signaling in relation to the enhanced contact with co-cultured cells. RESULTS: Both materials were biocompatible, translucent, and permeable, as assessed by metabolic assays, cell staining, and microscopy. While collagen scaffolds featured easy manipulation, washability, transferability, and biodegradability, CMC-PEG was advantageous for its easy preparation process and low variability in the number of accommodated cells. Both materials promoted cell-to-cell and cell-to-matrix interactions due to the scaffold structure and generation of cell aggregates. The metabolic activity of CLL cells cultured in CMC-PEG gel was similar to or higher than in conventional culture. Compared to the conventional culture, there was (i) a lower expression of VCAM1 in both materials, (ii) a higher expression of CCL4 in collagen scaffolds, and (iii) a lower expression of CXCR4 and MCL1 (transcript variant 2) in collagen scaffolds, while it was higher in a CMC-PEG gel. Hence, culture in the material can suppress the expression of a pro-apoptotic gene (MCL1 in collagen scaffolds) or replicate certain gene expression patterns attributed to CLL cells in lymphoid organs (low CXCR4, high CCL4 in collagen scaffolds) or blood (high CXCR4 in CMC-PEG).

• MeSH
• buněčné kultury metody   MeSH
• chronická lymfatická leukemie * patologie   metabolismus   MeSH
• gely chemie   MeSH
• kolagen * chemie   farmakologie   MeSH
• lidé MeSH
• polyethylenglykoly * chemie   MeSH
• receptory CXCR4 metabolismus   MeSH
• sodná sůl karboxymethylcelulosy * chemie   farmakologie   MeSH
• techniky 3D buněčné kultury metody   MeSH
• tkáňové podpůrné struktury * chemie   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH