The research performed by the authors was supported in part by grants from the European Union Seventh Framework Programme (FP7/2007- 2013) under Grant Agreement No. 229239, and from the AO Foundation, Exploratory Research Collaborative Research Program Acute Cartilage Injury/lesion/Defect (CRP ACI) under the project Bioactive and Biomimetic Scaffolds for Cartilage Regeneration (BIOCART).

1. Self-Assembling Peptide Scaffold (SAPS) Preparation
<ol>
	<li>Prepare a 0.5% solution of peptide RAD16-I in 20% (w/v) sucrose, in a micro-centrifuge tube. Sonicate the peptide solution for 20 min in an ultrasonic bath at maximum power and at room temperature (RT) to ensure homogeneous mixing. 
	NOTE: The volume of solution to be prepared will depend on the number of encapsulations desired. RAD16-I peptides do not suffer degradation under these sonication conditions.</li>
	<li>Dilute the peptide solution prepared in step 1.1 with the necessary volume of 10% (w/v) sucrose to obtain the desired final concentration. 
	NOTE: An important factor to take into consideration is the adjustment of the initial peptide concentration since the final matrix stiffness will depend on this parameter. The RAD16-I peptide solution should be 2x more concentrated than the desired final concentration of the 3D construct. Normally, the final RAD16-I peptide concentrations used are between 0.15&#8211;0.5% (w/v).</li>
	<li>Place the micro-centrifuge tube containing the peptide solution into an ultrasonic bath at RT for 20 min to ensure homogeneous mixing. 
	NOTE: The peptide solution, if not used immediately, can be stored for several months at 4 &#176;C.</li>
</ol>
2. Cell Suspension Preparation
<ol>
	<li>Centrifuge the cell suspension (obtained after trypsin treatment) at 60 x g and RT for 5 min.</li>
	<li>Remove the supernatant using a Pasteur pipette connected to a vacuum line and suspend the cell pellet in 3&#8211;5 mL of 10% (w/v) sucrose using a micropipette. Proceed to count cells using a manual or automatic cell counter. 
	NOTE: The 10% (w/v) sucrose solution is an isotonic and non-ionic medium that allows for cell maintenance and avoids peptide gelation during the mixing process.</li>
	<li>Centrifuge the cell suspension at 60 x g and RT for 5 min. Remove the supernatant and suspend the cell pellet again in the necessary volume of 10% (w/v) sucrose to obtain double the desired final cell concentration (recommendable approximately 4 x 106 cells/mL).</li>
	<li>Place a desired number of tissue culture inserts in each well of a 6-well plate (one insert per encapsulation).</li>
	<li>Sonicate the peptide solution prepared in step 1 at maximum power and RT for 5 min.</li>
	<li>Pipette and place 40 &#181;L of the cell suspension (obtained in 2.3) for each encapsulation desired plus approximately 10&#8211;15% of extra volume into a standard micro-centrifuge tube.</li>
</ol>
3. 3D Cell Encapsulation into SAPS
<ol>
	<li>Mix an equal volume of the RAD16-I solution in 10% sucrose (w/v) (40 &#181;L per encapsulation) with an equal volume of the cell suspension (40 &#181;L per encapsulation) prepared in step 2 to obtain a suspension of cells in the desired peptide concentration (in 10% sucrose). Mix by pipetting slowly up and down approximately 10 times. Avoid bubble formation during mixing. 
	NOTE: This step is critical and must be performed very carefully since cells are under hostile conditions due to the low pH peptide (approximately 3.5&#8211;4.0).</li>
	<li>Using a micropipette, carefully load 80 &#181;L of the suspension into each insert.</li>
	<li>Add 0.5 mL of culture media to the bottom of the well to wet the insert membrane, allowing media diffusion to promote peptide self-assembly and hydrogel formation. Let the peptide gel for approximately 20 min in the flow cabinet. 
	NOTE: The manipulation of the samples during setting time could end in gel breaking.</li>
	<li>Very slowly add 40 &#181;L of medium to the insert and let it slide down the inner wall to the gel. 
	NOTE: Normally, the loading of this volume should take between 20&#8211;30 s.</li>
	<li>Place the plate in the incubator (37 &#176;C, 5% CO2, humidified atmosphere) for 20 min. This step and successive medium additions will favor leaching of the sucrose and equilibration of cells with culture medium.</li>
	<li>Add 60 &#181;L of medium to the inner wall of the insert, allowing it to flow down the wall. Aspirate the medium of the well, which is rich in sucrose, and add 0.5 mL of fresh medium.</li>
	<li>Add 60 &#181;L of medium slowly to the insert and place the plate in the incubator (37 &#176;C, 5% CO2, humidified atmosphere) for 10 min.</li>
	<li>Aspirate the medium of the well and add 2.5 mL of fresh medium into the well. Add 200 &#181;L of medium into the insert. Place the plate in the incubator (37 &#176;C, 5% CO2, humidified atmosphere) to maintain the cells under the appropriate conditions.</li>
	<li>Change the medium every day. Remove the old medium from the bottom of the well. Add 2.5 mL of fresh medium to the well and 200 &#181;L to the insert. 
	NOTE: If cell differentiation continues, the specific inducing media will be added to the cultures, resulting in the desired cell type commitment (see below, Cell differentiation).</li>
</ol>
4. Cell Viability in 3D-SAPS
<ol> 
	<li>NOTE: The cell viability assay is known as the Live/Dead &#8212; Viability/Cytotoxicity assay as well.</li>
	<li>In a 15 mL tube, prepare the necessary volume of a fresh solution of 2 &#181;M calcein AM and 2 &#181;M of ethidium homodimer-1 (EthD-1) in PBS by vortexing for 10 s. Keep in the dark. 
	NOTE: The volume to be used for each sample will depend on the well size according to the following: 2 mL for each well of a 6-multiwell plate; 1 mL for each well of a 12-multiwell plate and 0.5 mL for each well of a 24-multiwell plate.</li>
	<li>Using a micropipette, rinse the 3D-SAPS samples 3 times with 2 mL of PBS, and then, cover it with the solution prepared in step 4.1. Incubate it at RT for 40 min in the dark.</li>
	<li>Using a micropipette, rinse the constructs with 2 mL of PBS 5 times.</li>
	<li>Visualize the samples under a fluorescence microscope using 10X or 20X magnification.</li>
</ol>
5. Cell Differentiation
<ol>
	<li>Culture cells (37 &#176;C, 5% CO2, humidified atmosphere) in Chondrocyte Basal Medium (CBM) with growth supplements. 
	NOTE: The preparation of the medium is described in the manufacturers&#39; instructions (See Materials Table).</li>
	<li>Induce cell differentiation at day 2 with chondrogenic medium containing recombinant human transforming growth factor-b1 [TGFb1] (10 ng/mL), L-Ascorbic Acid 2-phosphate [AA2P] (25 &#181;g/mL) and Dexamethasone (100 nM).</li>
	<li>Maintain the culture for 4 weeks in an incubator with a humidified atmosphere at 37 &#176;C and 5% CO2. Change chondrogenic medium every other day.</li>
</ol>
6. Toluidine Blue (TB) Staining
<ol>
	<li>Wash the 3D-SAPS cultures twice by adding and then removing 2 mL of PBS with a micropipette. Fix them by adding 2 mL of 2% (w/v) p-formaldehyde in PBS for 2 h at RT.</li>
	<li>Wash twice with 2 mL of 0.1% Triton X-100 in PBS (PBST).</li>
	<li>Incubate with 2 mL of 0.05% TB in water for 20 min at RT.</li>
	<li>Wash several times (at least 3 times) with 2 mL of PBS until the PBS solution removed is clear.</li>
	<li>Analyze samples by visual inspection under a stereoscopic microscope11,13,14. 
	NOTE: TB forms complexes with anionic glycoconjugates at the extracellular matrix, such as proteoglycans (PG) and glycosaminoglycans (GAG), which are mainly secreted by chondrocyte-like cells. Positive samples are stained blue.</li>
</ol>
7. Western Blot
NOTE: The procedure used in this protocol was previously described in reference11.
<ol>
	<li>Lyse 3D-constructs in RIPA buffer containing protease inhibitor cocktail by pipetting.</li>
	<li>Prepare acrylamide gel according to gel-volume requirements (of the specific gel running equipment) and run the cell lysates for 90 min at 150 V16. 
	NOTE: Acrylamide gel is usually prepared at a concentration of 7.5&#8211;10% (w/v), depending on the protein&#39;s molecular weight.</li>
	<li>Transfer the proteins contained in the gel into a polyvinylidene difluoride (PVDF) membrane by applying 40 V for 2 h at RT.</li>
	<li>Incubate the PVDF membrane at RT for 2 h in blocking buffer (4% (w/v) nonfat powdered milk in PBST) in a separate container with enough volume to cover the membrane.</li>
	<li>Incubate the membrane at RT for 1 h with primary antibodies at a final concentration of 1 mg/mL in PBST. See Materials Table.</li>
	<li>Add secondary antibodies at a concentration of 1 mg/mL. See the Materials Table.</li>
	<li>Prepare the membrane for hP detection with a Chemiluminescent substrate16. See Materials Table.</li>
	<li>Take chemiluminescent images16.</li>
</ol>

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The authors have nothing to disclose.

Previously, our group and others have described the use of three-dimensional (3D) culture platforms with diverse cell systems3,4,5,6,7,8,9,10,11,12,13,14,15. In the present work, we describe an easy and reliable method of obtaining 3D culture systems that are applicable to any type of mammalian cells including any type of functional cell, embryonic or adult stem cells, or eventually, dysfunctional cells isolated from biopsies or tumors, and so on. In addition, independently, if stem cells are of embryonic or adult origin they would have a better lineage commitment capacity in the 3D environment than in classical 2D culture dishes10,11,12,13,14,15. Therefore, the cell culture in this system would lead to differentiation into functional tissue-like structures that could be used in different applications, from reparative or regenerative biomedicine to toxicological and pharmacological platforms.
We have shown a clear gain in cell function because the cellular microenvironment is similar to that of natural tissues in terms of the matrix structure and biomechanical, biophysical and biological parameters. Nevertheless, as the system becomes more complex, the number of parameters that need to be regulated also increases, which includes the need for an external supporting platform (such as an active perfusion system to avoid mass transfer phenomena-associated issues). Three-dimensionally cultured cells perform better in terms of regulating essential activities, such as migration, proliferation and differentiation. They could form complex networks allowing enhanced cellular crosstalk, which is by far an essential consequence of growing and differentiating in 3D. The fact that SAPS could create conditions in vitro similar to those extracellular matrix proteins represents an advantage since a rational study of the effect produced for each component added to the scaffold (growth factor, polysaccharide or signaling peptide) could be easily carried out.
The clear advantages of the use of SAPS compared to other natural scaffolds, such as collagen type I and Matrigel, are the following: 1) SAPS is a synthetic biomaterial with minimal variation from batch to batch production; 2) SAPS has the capacity to be functionalized with specific peptide motifs; and 3) SAPS presents low biodegradability in vitro, which permits the maintenance of the 3D construct with the same biophysical, biomechanical and structural properties over time. However, the limitation of using SAPS versus other scaffolds is found during the encapsulation step, where cells are in a hostile milieu due to the low pH. Therefore, this is a critical step in the described methodology. Moreover, it is important to set the SAPS concentration for each specific cell type before starting any experiment. This is, in fact, essential when cells are cultured on or into these biomaterials, since each particular cell type will present optimal biomechanical growth conditions.
Finally, we believe that the design and fabrication of this type of biomaterial scaffold would enhance the development of more physiological and reliable 3D tissue models to help the pharmaceutical industry to develop better therapeutic approaches for regenerative medicine, cancer or any medical treatment.

For many decades, mammalian cell culture has been performed under experimental conditions using classical two-dimensional (2D) culture systems due to practical and economic issues regardless of the non-physiological aspects. Although this culture system helps to study and understand most molecular and cellular mechanisms, we know today that new cell culture paradigms are needed to study more complex cellular systems. Therefore, three-dimensional (3D) culture systems are needed to recreate a microenvironment that is biophysically, biomechanically and biologically more similar to that of natural tissues. In recent years, 3D culture systems, in general, have become more prevalent among researchers and industry since they represent a new model of study or screening in which cells can grow in space, create cell-to-cell or cell-to-matrix interactions, migrate and eventually differentiate into specific cell lineages.
The overall goal of this methodology is to recreate an in vitro cellular microenvironment that is closer to the in vivo microenvironment. In particular, the synthetic self-assembling peptide scaffold (SAPS) is a type of biomaterial with unique properties; it forms a network of nanometer-sized pores made out of weak interactions among peptides with mechanical and structural properties similar those of natural extracellular matrices. In other words, the rationality behind the use of this material is that it creates a truly 3D-environment that is ideal for obtaining pseudo-3D tissues or organ units. However, most importantly, the 3D context allows the 3D structure to gain new biological functions that normally are not present in 2D culture platforms, such as properties related to tissue architecture, mass transfer phenomena, cell patterning and eventually tissue morphogenesis, which are key factors in future research and development of functional tissues and organs1,2. Moreover, an advantage of SAPS over their natural counterparts (Collagen, Matrigel) is that they are very stable at room temperature and do not require special conditions for post-production, distribution or storage3,4,5,6,7,8,9,10,11,12,13,14,15. SAPS is easy to handle, when desired; 3D gels can be simply obtained by increasing the ionic strength or by adjusting the pH to neutrality1,2. Finally, the methodology described here has been extensively used in vitro to promote maintenance, growth, and differentiation of a number of cell types, including chondrocytes, hepatocytes, endothelial cells, osteoblasts, neuronal cells as well as embryonic and somatic stem cells3,4,5,6,7,8,9,10,11,12,13,14,15. In the present work, we describe the use of a 3D-culture system to differentiate human expanded articular chondrocytes (hACh) into cartilage-like tissue as previously described11.
Here, a method to culture cells in a 3D system using SAPS is described. In this synthetic biomaterial, cells are first mixed with a peptide solution, which is subsequently induced to self-assemble, creating a network of nanometric dimensions around the cells and therefore creating a truly 3D environment (Figure 1). It is important to consider that cellular behavior is affected by matrix stiffness values (i.e., proliferation, migration and differentiation). Therefore, a common methodological control is to culture cells on top of the peptide scaffold with the same stiffness values (culture on two dimensions).

<table><tbody><tr><td>Human articular chondrocytes (Ach)</td><td>Lonza</td><td>CC-2550</td><td></td></tr><tr><td>RAD16-I peptide solution (PuraMatrix)</td><td>Corning</td><td>354250</td><td></td></tr><tr><td>Sucrose (tissue culture grade)</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Cell culture inserts (0.4 &amp;micro;m pore, 12 mm diameter)</td><td>Millipore</td><td>PICM01250</td><td></td></tr><tr><td>Chondrocyte Basal Medium (CBM)</td><td>Lonza</td><td>CC-3217</td><td></td></tr><tr><td>SingleQuots of Growth Supplements</td><td>Lonza</td><td>CC-4409</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Lonza</td><td>DE14-801F</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D8893</td><td></td></tr><tr><td>L-ascorbic acid 2-phosphate (AA2P)</td><td>Sigma</td><td>A8960</td><td></td></tr><tr><td>Human transforming growth factor-&amp;beta;1 (TGF-&amp;beta;1)</td><td>Millipore</td><td>GF111</td><td></td></tr><tr><td>RIPA buffer</td><td>Sigma</td><td>R0278</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche</td><td>11836153001</td><td></td></tr><tr><td>Polyvinylidene difluoride (PVDF) membrane</td><td>Invitrogen</td><td>LC 2005</td><td></td></tr><tr><td>SuperSignal West Pico Chemiluminescent Substrate</td><td>Thermo Scientific</td><td>34080</td><td></td></tr><tr><td>Anti-Actin</td><td>SCBT</td><td>sc-1615</td><td></td></tr><tr><td>Anti-Collagen I</td><td>Abcam</td><td>ab138492</td><td></td></tr><tr><td>Anti-Collagen II</td><td>Abcam</td><td>ab3092</td><td></td></tr><tr><td>Anti-Collagen X</td><td>Abcam</td><td>ab182563</td><td></td></tr><tr><td>Antigoat IgG-HRP</td><td>Abcam</td><td>ab97100</td><td></td></tr><tr><td>Anti-mouse IgG-HRP</td><td>Abcam</td><td>ab97023</td><td></td></tr><tr><td>Anti-rabbit IgG-HRP</td><td>Abcam</td><td>ab97051</td><td></td></tr></tbody></table>

culturing mammalian cells in three-dimensional peptide scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

In the present work, a simple and faster protocol to culture cells in 3D using SAPS is described to obtain reliable qualitative and quantitative data. SAPS creates a 3D microenvironment with unique properties that allow cultured cells under desired conditions to proceed to cell maintenance, proliferation and/or differentiation3,4,5,6,7,8,9,10,11,12. In our group, several studies have been performed using human adipose tissue derived progenitor cells (hAPC), expanded human articular chondrocytes (hACh) and human normal dermal fibroblasts (hNDF) to culture, expand and differentiate them into cartilage-like tissue10,11,12. Here, we describe the 3D culture and re-differentiation of expanded hACh into cartilage-like tissue, as previously described11. Hence, we encapsulate cells in 3D-SAPS and culture them in chondrogenic induction medium, as described in Figure 1 (and in the Protocol). After 24 h, samples were assessed for viability with the Live/Dead Viability/Cytotoxicity Assay. The results indicate that a few percent of cells were dead (red) after 5 days of encapsulation and almost no dead cells were observed after 4 weeks of culture (Figure 2). Next, we stained them for glycosaminoglycan (GAG) deposition at the extracellular matrix using the Toluidine blue staining method. As expected, the induction group responded properly to the treatment, showing strong staining (Figure 2).
Finally, molecular markers of chondrogenesis and hypertrophy markers, including collagen type I, II and X, were analyzed by western blot (Figure 3). Collagen type I (COL1) was observed in the 2D and 3D systems but a lower band of ~130 kDa (representing the processed mature COL1 protein to be assembled at the extracellular matrix) was only observed in 3D. This result clearly indicates that cells undergoing differentiation in three dimensions do proceed in a more physiological way. Remarkably, collagen type II (COL2) was detected - as a single band of the expected molecular weight present at the extracellular matrix - only in 3D constructs cultured under chondrogenic conditions. Finally, collagen type X (COL10) was detected in both 2D and 3D constructs cultured under chondrogenic conditions, indicating some degree of hypertrophy after 4 weeks of culture.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57259/57259fig1v2.jpg" /> 
Figure 1: Schematic representation of cell encapsulation into 3D-SAPS. Schematic representation of the hACh encapsulation is described to obtain cells randomly dispersed within the 3D-SAPS. 1. Self-assembling peptide scaffold (SAPS) preparation; 2. cell suspension preparation; and 3. 3D-cell encapsulation into SAPS. Note that the schematic only shows steps 3.1 to 3.4.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57259/57259fig2.jpg" /> 
Figure 2: Cell performance and chondrogenic differentiation in 3D-SAPS cultures. Cell viability staining performed at 5 days and 4 weeks of culture detected under a fluorescent microscope (live cells in green and dead cells in red). Scale bars = 200 &#181;m. General chondrogenesis commitment assessed by Toluidine blue staining (sulfated glycosaminoglycans in blue) of 3D-SAPS constructs cultured in control and chondrogenic media. Scale bars = 500 &#181;m. This figure has been modified from L. Recha-Sancho et al.11. <a href="/files/ftp_upload/57259/57259fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57259/57259fig3.jpg" /> 
Figure 3: Collagen I, II and X expression of hACh undergoing chondrogenesis. hACh cultured in a 2D-monolayer and in 3D-SAPS after 4 weeks in chondrogenic media and controls were assessed for the expression of collagen type I (COL1), collagen type II (COL2) and collagen type X (COL10). Actin expression was used as an internal control. This figure has been modified from L. Recha-Sancho et al.11. <a href="/files/ftp_upload/57259/57259fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds at JoVE.com

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

culturing-mammalian-cells-in-three-dimensional-peptide-scaffolds

Research

JoVE Journal

Bioengineering

10.1K Views.  Ramon Llull University. Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Video: Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Cancer Research

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Medicine

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

Advanced Two-Layered 3D Co-Culture System for Epithelial and Fibroblast Cells

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

Author Spotlight: Advancing 3D Coculture Systems with PVA-PCL Nanofibrous Membranes 

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and tunable 3D hydrogel, suitable for culturing cells and tissue in a setting that matches their native environment. This is particularly important for researchers investigating the initiation, growth, and treatment of cancer where the interaction between cells and their local extracellular matrix is a fundamental part of the model. Moving to 3D culture can be challenging and is often associated with a lack of reproducibility due to high batch-to-batch variation in animal-derived 3D culture matrices. Similarly, handling issues can limit the usefulness of synthetic hydrogels. In response to this need, we have optimized a simple self-assembling peptide gel, to enable the culture of relevant cell line models of cancer and disease, as well as patient-derived tissue/cells. The gel itself is free from matrix components, apart from those added during encapsulation or deposited into the gel by the encapsulated cells. The mechanical properties of the hydrogels can also be altered independent of matrix addition. It, therefore, acts as a &#8216;blank slate&#8217; allowing researchers to build a 3D culture environment that reflects the target tissue of interest and to dissect the influences of mechanical forces and/or biochemical control of cell behavior independently.

We present a method for creating a 3D cell culture environment, which can be used to investigate the importance of cell/matrix interactions in cancer progression. Using a simple self-assembling octapeptide, the matrix surrounding encapsulated cells can be controlled, with independent regulation of mechanical and biochemical cues.

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and ...

Biology

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

Summary

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Chapters in this video

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications