Human biopsies in nanofibrillar cellulose hydrogel – a novel method for long-term tissue culture; Niklander, Johanna; Rinner, Beate

article

Human biopsies in nanofibrillar cellulose hydrogel – a novel method for long-term tissue culture

By Dr Beate Rinner (Medical University of Graz), Johanna Niklander (Medical University of Graz)

20 December 2021

Three-dimensional (3D) biopsy culture models could save the drug discovery industry time and money. In this article, Johanna Niklander, University of Helsinki and Medical University of Graz, and Dr Beate Rinner, Medical University of Graz, outline a recent study they took part in, investigating long-term 3D tissue cultures from human biopsy samples to evaluate the preservation of tissue-typical protein expressions.

Introduction

Most of the current in vitro cell models consist of one or two cell types, whereas to be physiologically relevant, the model should also take into account the complex cellular interactions within the tissue microenvironment.^1,2 However, highly complex in vitro models are laborious^3,4 and may accumulate unintentional design errors.⁵ Culture materials for 3D growth have also been known to affect the diffusion environment of experiments,⁶ introducing xenofactor contaminations to results^7,8 and affecting cell behaviour via scaffold stiffness or growth factors.^9,10

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Formation of physiological structures that require growth in cell aggregate size also face limitations due to limited nutrient and oxygen diffusion in 3D cell cultures.^11-13 Due to numerous challenges in reconstructing disease models in vitro, ex vivo samples have been gaining solid ground as experimental material. Intact biopsy pieces are easy and fast to process for cultivation, readily contain all cell populations of the studied biological target and have the genetic makeups of patients. When reliably validated for general use, the robustness of setting up a 3D biopsy culture model could significantly save time, costs and resources in the industry.

The greatest challenge when using tissue sample cultures is to sustain all relevant cell types long term in culture conditions.¹¹ In this study, our aim was to establish long-term tissue cultures from human biopsy pieces and to evaluate the preservation of tissue-typical protein expressions. Experimental human biopsy samples were acquired from healthy skin (HS), healthy foreskin (FRSK) and glioblastoma multiforme (GBM) tissue donors. Ethical approval was in accordance with the ethical standards of the responsible committee for human experimentation and the current version of the Declaration of Helsinki. To qualify the experimental culture model also for future clinical prospects, we utilised animal-free nanofibrillar cellulose (NFC) hydrogel as a culture scaffold.^14-16 Wood-derived, biocompatible NFC is composed of cellulose fibrils of nanoscale diameter and microscale length and the fibril size and viscoelastic properties resemble those of an extracellular matrix (ECM).¹⁷ To observe how NFC compares to a commonly known animal‑derived culture scaffold, parallel biopsy cultures were grown with Engelbreth‑Holm‑Swarm (EHS) mouse sarcoma basement membrane extract gel.^10,18

Figure 1: Experimental setup
(A) HS, FRSK and GBM biopsies were treated with antibiotics and dissected
(B) Dissected biopsy pieces were implanted as flat tissue layers into 96-culture wells holding 50µl layers of NFC or Matrigel scaffold and were covered by another 50µl layer of the same scaffold
(C) 130µl of culture medium was added on top of the uppermost scaffold layers and was refreshed with 100-120µl culture medium every three days. Culture morphology development was observed with a light microscope on days one, six, 12, 18 and 27. Biopsy pieces were collected for histological analysis during ex vivo biopsy dissection (d0) and from 3D culture wells on days three, six, 12 and 28.

Patient biopsies were dissected and inserted to NFC and Matrigel scaffolds (Figure 1). Morphological development of the cultures was monitored with light microscopy and histological correspondence with original ex vivo biopsy compositions was examined with immunohistochemical (IHC) profiling. Normal skin and foreskin tissues were evaluated for cytokeratins 5 and 6 (CK 5/6), epidermal E-cadherin and mesenchymal vimentin in order to evaluate tissue structure survival. Glioblastoma samples were evaluated for typical histopathological disease profile using the Olig2, S100, Nestin, NOTCH1, MAP2 and GFAP biomarkers.

Results

HS biopsies appeared relatively static during NFC cultivation. Early rearrangements in the biopsy shape and modest cell spreading could be observed in Matrigel culture. NFC-cultivated FRSK tissues showed growth of tissue mass, development in the biopsy shape and signs of protruding or degrading structures. When cultivated in Matrigel scaffolding, rapid cell spreading, tissue growth and biopsy unfolding were observed. No noticeable amounts of dying cells or other debris were detected with either skin tissue types after the first three days of culture in NFC or Matrigel. GBM cultures showed greatest variation in the growth rates of individual culture wells and most prominent size reductions in cultivated tissue pieces.

Figure 2: Morphology development of cultivated human GBM biopsy pieces. Reductions in biopsy piece sizes were common during NFC cultivation (A-D). New growth appeared on the earliest on day six in both scaffolds and consisted of protruding structures (I: NFC culture presented). Tissue pieces showed rapid spreading growth in surrounding Matrigel scaffolding, causing reductions to the original sample size (E-H). Observed novel growth consisted of protruding cells in both scaffold types (J: NFC; K: Matrigel). Figure scale bars 200µm (A-H), 100µm (I, J), 50µm (K).

Typical morphology development during both NFC and Matrigel cultivation included partial degradation of original biopsy structures (Figure 2A-D) and appearance of protruding cells (Figure 2I, J, K). Extensive cell spreading in 3D Matrigel correlated with biopsy size reductions (Figure 2E-H).

Histological analysis of GBM cultures revealed sustained ex vivo protein expressions and novel expression patterns

Analysis of GBM cultures revealed spindle shaped, highly pleomorphic tumour cells in ex vivo, NFC and Matrigel (Figure 3A, E, I). Antibody analysis revealed a sustaining ex vivo protein expression profile in all culture conditions until day 28. Immunoreactivity to oligodendrocyte transcription factor 2 (Olig2) had evenly distributed nuclear stain from ex vivo to day 28 in vitro (Figure 3B, F, J). Similarly, calcium ion binding protein B (S100) detection remained stable from ex vivo to day 28 in vitro (Figure 3C, G, K). Neuroectodermal stem cell – intermediate filament VI (Nestin) appeared during cultivation and was detected in cultivated day 28 samples (Figure 3D, H, L).

Discussion and conclusions

In this study, we developed a technically efficient methodology to sustain intact and heterogeneous human tissues long-term in vitro. Human skin and brain tissue samples sustained their histological profiles for 28 days when cultivated in NFC and Matrigel matrices and these culture methods enabled constant morphology and histology characterisation. The suitability of NFC in tissue culture enables the use of species‑different substances to be reduced in experimental settings.

Figure 3: Histological analysis of GBM ex vivo (day 0) and cultivated biopsy pieces (day 28). Analysis revealed pleiomorphic tumour cell structure in ex vivo and cultivated day 28 GBM pieces (A, E, I). Olig2 showed equally distributed detection stain in all samples from ex vivo to NFC and Matrigel cultivated day 28 GBM samples (B, F, J). S100 was similarly detectable already in the ex vivo biopsy piece and remained stable during cultivation in both culture conditions (C, G, K). Nestin was absent in ex vivo sample (D) and appeared during cultivation in both culture conditions (H, L). Figure scale bars 100µm.

GBM patient tissue was utilised as the neoplastic biopsy model. Analysis revealed that typical histopathological GBM tumour tissue structure was sustained during NFC and Matrigel cultivation. The ex vivo-, NFC- and Matrigel-cultivated glioblastoma biopsy pieces stained positive for the characterised Olig2, S100 and Nestin and additionally also NOTCH1, MAP2 and GFAP biomarkers (data not shown), indicating successfully preserved brain tumour tissue in vitro. According to the acquired profiling, the biopsy pieces contained neoplastic glial cells or mobile immature glial cells expressing MAP2, typical for structural stabilisation during neuronal development;¹⁹ mature astrocytes or glial astrocytes (GFAP),^20,21 expressing S100 concentration-dependent proliferation, neurite outgrowth or inflammatory- and neoplasia-related activity^22,23 and stem cells, indicated by Olig2 that has been reported to sustain brain tumour stem cell populations.²⁴ The expressions of general upstream-regulator NOTCH1 and Nestin are also common in brain neoplasia, where NOTCH1 maintains and drives the proliferation of cancer stem cells²⁵ and Nestin marks differentiating neural progenitor cells.²⁶ In addition, NFC-cultivated glioblastoma tissues showed possible signs of reactive gliosis, observed as peripheral staining patterns of NOTCH1, MAP2 and GFAP in the biopsy pieces.^27-29 In non-neoplastic tissues, gliosis is triggered by tissue trauma factors, such as growth factors released from damaged tissue, factors presented by direct blood contact and descending oxygen and nutrient levels after cutting off blood circulation.³⁰

The NFC culture environment appeared consistent in preserving the histopathological traits of patient biopsies. The observed cell outgrowth and developments in tissue morphology also indicate culture viability. When comparing the two culture scaffolds, the 3D Matrigel appeared to promote cell migration at the expense of tissue integrity in FRSK and GBM cultures, which was also marked by the losses of tissue heterogeneity and typical gliosis processes in GBM histology. Although often considered the golden standard in many 3D culture applications, the use of Matrigel has been disputed due to growth-guiding and cancerous growth factors in experimental settings,^31-33 as well as the fact that laminin-111 is crucial mostly during tissue development and expressed only in few tissue epithelia in adults.³⁴

We consider the combination of biopsy tissue and NFC hydrogel a very promising 3D model, since NFC matrix ensures that no unknown growth-affecting factors arise from the scaffold material and that the detected animal proteins originate exclusively from the cultured cells or known medium supplements. Long-term NFC cultivation of human samples could therefore be used for various research questions, such as modelling different disease models, drug screening or personalised medicine.

This study is in preprint in bioRxiv.

Acknowledgements

This work was supported by UPM-Kymmene, Finland. We thank the many researchers and staff in the Core Facility Alternative Biomodels and Preclinical Imaging and Diagnostic and Research Institute of Pathology at the Medical University of Graz for their extra time and effort given during the methodology development.

About the authors

Johanna Niklander has an MSc in Genetics with a developmental biology major. She completed her thesis at the University of Helsinki, where she characterised NFC culture matrix with a non-neoplastic breast cell line. She continued her research in the Medical University of Graz, where she characterised the use of NFC for the cultivation of human patient tissues. Johanna graduated as an Azure Cloud Developer in 2021 and is currently working with data warehousing and data processing in Avanade Finland.

Dr Beate Rinner is Associate Professor at the Medical University of Graz, Head of the Core Facility Alternative Biomodels and Preclincial Imaging and founder of the Cellbank Graz. Her research focuses on the establishment of rare tumour cell lines and the generation of autologous tumour models to advance personalised medicine and to identify and understand important signalling pathways. Additionally, cell communication and biomarker development through the release of extracellular vesicles are among her areas of expertise.

CONTRIBUTORS:

Raili Koivuniemi – University of Helsinki

Alexander Stallinger – Medical University of Graz

Florian Kleinegger – Medical University of Graz

Lauri Paasonen – UPM Biomedicals

Silke Schrom – Medical University of Graz

Bernadette Liegl-Atzwanger – Medical University of Graz

Iris Zalaudek – University of Trieste

Gord von Campe – Medical University of Graz

Georg Singer – Medical University of Graz

Johannes Haybaeck – Medical University of Graz

Marjo Yliperttula – University of Helsinki

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