Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.
Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.
Centralnervesystemet (CNS) kan påvirkes af en række lidelser, der involverer vaskulære, strukturelle, funktionelle, infektiøs eller degenerativ. Det anslås 6,8 millioner mennesker dør hvert år som følge af neurologiske lidelser, der udgør en stigende samfundsøkonomisk byrde på verdensplan 1. Det er dog kun få af de lidelser har tilgængelige behandlinger. Der er derfor et kritisk behov for innovative terapeutiske strategier for patienter, der lider af neurologiske lidelser. Desværre er der mange CNS-målrettede lægemidler mislykkes i kliniske forsøg, delvis på grund af udnyttelsen af utilstrækkelige prækliniske forskning modeller, som ikke tillader vurdering af akutte og kroniske virkninger med fysiologisk relevante funktionelle udlæsninger.
Trods betydelige forskningsindsats i løbet af de seneste årtier, er der et stort beløb ukendt om strukturen og funktionen af CNS. For at få mere viden, dyremodeller ofte osed til at modellere patologiske tilstande, såsom traumatisk hjerneskade (TBI) eller demens, især i prækliniske studier. Adskiller sig imidlertid væsentligt fra dyr mennesker både anatomi CNS, samt i funktion, genekspression og metabolisme 2-4. På den anden side, 2D in vitro kulturer er den fælles metode til at undersøge cellebiologi og anvendes rutinemæssigt til lægemiddelforskning. Men 2D cellekulturer mangler kompleksitet og fysiologiske relevans i forhold til menneskelige hjerne 5-7. Mens der er ingen erstatning for de lave omkostninger og enkelhed i cellekultur studier 2D eller kompleksitet, som dyremodeller, kunne 3D tissue engineering generere forbedrede forskning modeller til at lukke hullet, der eksisterer mellem 2D in vitro og in vivo tilgange. 3D tissue engineering giver mere fysiologisk relevante forsøgsbetingelser opnået af 3D celle-celle interaktioner og ekstracellulære signaler fra de biomateriale stilladser. DespITE den betydelige beviser bag værdien af 3D-kulturer, der i øjeblikket kun få 3D CNS væv modeller som stamcelle-afledte organoide kulturer 8-10, neurospheroids 11 og spredte hydrogel kulturer 12,13. Der er blevet anvendt avancerede tekniske metoder, herunder flerlags litografi 14 og 3D-print 15 for at studere lunge-, lever- og nyrevæv. Der er imidlertid manglen på 3D CNS modeller, der giver mulighed for opdelte neuronal vækst, såsom efterligne den korticale arkitektur og biologi. Adskilt væksten af neuritter fra neuronale cellelegemer er tidligere blevet påvist i 2D kulturer ved hjælp microfabrication 16,17 tillader studiet af axon-tarmkanalen opsporing, calciumindstrømning, netværksarkitektur og aktiviteter. Denne idé inspireret os til at udvikle en 3D-polariseret nervevæv, hvor celle organer og axonale skrifter er placeret i forskellige rum efterligner den lagdelte arkitektur af hjernen 18 </sup>. Vores tilgang er baseret på anvendelsen af unikke silke stillads design, som kan rumme høj tæthed af celler i et lukket volumen og tillader udvækst af tætte axonal netværk i en kollagengel. Her demonstrerer vi den fulde samling proceduren for hjerne-lignende væv, herunder stillads fabrikation og neuronal kultur.
Here we described the guidelines to assemble a compartmentalized 3D brain-like tissue model. The model is characterized by dense polarized culture of neurons resulting in the development of two morphologically different compartments: one containing densely packed cell bodies, second containing pure axonal networks. Overall, the scaffold architecture and growth pattern mimic brain cortical tissue including the six laminar layers and white matter tracts21.
The donut-shaped silk protein-based tissue model allows for modifications and tuning of mechanical properties, versatile structural forms, hydrogel fillers and cellular components. Thus, this tissue model establishes a base platform for a wide range of studies. Silk is a favorable candidate for biomaterial platforms due to its biocompatibility, aqueous-based processing, and robust mechanical and degradation properties22. The silk matrix also serves as a stable anchor to reduce collagen gel contraction over time in culture. The properties such as silk concentration and porosity of the scaffold used in this model have been previously adjusted to achieve optimal cell growth and mechanical properties resulting in brain-like tissue elasticity18,23,24. We suggest to keep these parameters constant to ensure the successful outcome and reproducibility of the experiments.
The 3D neuronal network formation was achieved by combining two types of biomaterials with different mechanical properties: a stiff scaffold to provide neuronal anchoring and a softer gel matrix to permit axon penetration and connectivity in 3D. Selective material preferences of the silk scaffold and soft hydrogel provide the underlying principle for compartmentalizing the neurons from the axonal connections. Due to the inert nature of the silk scaffold, functionalization with poly-D-lysine is required for neuronal attachment. However, other cell adhesion promoting factors can be applied such as RGD25 or fibronectin26. To achieve the 3D network formation the silk scaffold needs to be filled with hydrogel in a timely manner as soon as neurons are attached to the scaffold. Likewise, the collagen matrix filler can be replaced by other hydrogels, thus allowing the study of the influence of 3D extracellular environments on axonal network formation to serve as a platform to evaluate novel hydrogels in terms of neuronal compatibility. Additionally, apart from rat cortical neurons other neuronal sources such as hippocampal neurons or induced pluripotent cell (iPSc)-derived neurons can be utilized. Moreover, the tissue model can be used to study heterocellular interactions by including glial cells along with neurons and to build more complex brain-like tissue.
As shown previously, our model can be utilized to evaluate neuronal functionality in 3D microenvironment with a variety of assays such as cell viability, gene expression, LC-MS/MS and electrophysiology, thus demonstrating physiological relevance of the model18. Other methods, which are frequently utilized to evaluate 3D tissue-engineered constructs, such as immunostaining and microscopy8,9,11 can also be used to assess cell distribution and extent of axonal network formation. However, it has to be noted, that due to the high density of the collagen matrix, the penetration of antibodies and depth of the imaging is limited to few hundred micrometers. Moreover, the signal to noise ratio may be affected by nonspecific background fluorescence. This can be overcome by using lipophilic cell tracers and genetically expressed fluorescent proteins which diminish the need for immunostaining11. Alternatively, the imaging can be performed with 2-photon microscopy instead of usual one-photon technique, which may reduce the signal loss, photobleaching, and can be extended to several hundred micrometers of depth.
Summarizing, the silk and collagen-based brain-like tissue offers a robust platform to study neuronal networks in 3D and is a starting point for the development of more advanced models of neurological disorders in the future. Independent from the mode of evaluation, the relative simplicity of this tissue model supports its utility, success and reproducibility.
The authors have nothing to disclose.
We thank the laboratory of Dr. Stephen Moss for providing embryonic rat brain tissues. M.D.T. designed the original protocol. This work was funded by National Institutes of Health P41 Tissue Engineering Resource Center Grant EB002520. K.C. was supported with Postdoctoral Fellowship from German Research Foundation (DFG) (CH 1400/2-1).
Na2CO3 | Sigma-Aldrich | 223530 | – |
LiBr | Sigma-Aldrich | 213225 | – |
MWCO 3500 dialysis cassettes | Thermo Fisher | 66110 | – |
Heating plate | Fisher Scientific | Isotemp | – |
Centrifuge 5804 R | Eppendorf | – | – |
Sieve | Fisher Scientific | – | No. 270, No. 35, No. 30 |
PTFE mold | – | – | made in house (Figure 1) 10 cm diameter, 2 cm height |
NaCl | Sigma-Aldrich | 71382 | – |
Biopsy Punch 5mm | World Precision Instrument | 501909 | – |
Biopsy Punch 2mm | World Precision Instrument | 501908 | – |
poly-D-lysine | Sigma-Aldrich | P6407-5MG | final concentration 10 ug/ml, dissolved in water |
PBS | Sigma-Aldrich | D1283-500ML | – |
Trypsin | Sigma-Aldrich | T4049-500ML | – |
DNase | Roche | 10104159001 | final concentration 0.3% |
Soybean protein | Sigma-Aldrich | T6522-100MG | final concentration 1 mg/ml |
Neurobasal medium | Gibco | 21103049 | warm up to 37°C before use |
B27 supplement 50x | Gibco | 17504-044 | – |
Glutamax | Gibco | 35050-061 | – |
Penicilin Streptomycin | Corning | 30-002-CI | – |
Collagen I, rat tail, 100 mg | Corning | 354236 | final concentration 3 mg/ml |
NaOH | Sigma-Aldrich | S2770 | corrosive |
Propidium Iodide | Sigma-Aldrich | P4170-10MG | toxic |
Fluorescein Diacetate | Sigma-Aldrich | F7378-5G | – |
Olympus MVX10 | Olympus | – | – |
Paraformaldehyde | Sigma-Aldrich | P6148 | toxic, final concentration 4% |
Bovine Serum Albumin | Sigma-Aldrich | A7906-10G | – |
anti-βIIITubulin antibody | Sigma-Aldrich | T2200 | rabbit 1:500 |
anti-rabbit Alexa-546 | Molecular Probes | A11010 | goat 1:250 |
goat serum | Sigma-Aldrich | G9023-5ML | – |
Leica SP2 confocal microscope | Leica | – | objective 20x |
QIAshredder | Qiagen | 79654 | – |
AllPrep DNA/RNA/Protein Mini Kit | Qiagen | 80004 | – |
Ethyl alcohol, Pure | Sigma-Aldrich | E7023 | – |
2-Mercaptoethanol | Sigma-Aldrich | 63689 | |
NanoDrop 2000c/2000 UV-Vis Spectrophotometer | Thermo Scientific | – | – |
BCA protein assay | Thermo Scientific | 23225 | – |
Plate reader Spectramax M2 | Molecular Devices | – | absorbance 562 nm |
Micro Scissors, Economy, Vannas-type | TedPella | 1346 | – |