The authors would like to thank Norman Kr&uuml;ger for his excellent technical support.

1. Part 1: Culture of hNPCs in PuraMatrix
<ol>
	<li>In advance to the generation of a scaffold with a PuraMatrix concentration of 0.25% without laminin one needs to prepare the following solutions</li>
	<li>Prepare a solution containing 20% sucrose and a solution containing 10% sucrose dissolved in sterile distilled water.</li>
	<li>For solution 1 mix 120 &mu;l of the 20% sucrose solution with 120 &mu;l of distilled water in a 1.5 ml conical tube.</li>
	<li>For solution 2 mix 60 &mu;l of the PuraMatrix solution with 60 &mu;l of 20% sucrose solution in a 1.5 ml conical tube.</li>
</ol>
For a Preparation of a scaffold with a PuraMatrix concentration of 0.25% supplemented with laminin (8 &mu;g per 100 &mu;l Matrix) one needs to prepare the following solutions.
<ol class="lroman">
	<li>For solution 1 mix 72 &mu;l of distilled water with 120 &mu;l of 20% sucrose solution and 48 &mu;l laminin in a 1.5 ml conical tube.</li>
	<li>For solution 2 mix 60 &mu;l PuraMatrix solution with 60 &mu;l of the 20% sucrose solution in a 1.5 ml conical tube.</li>
</ol>
<ol start="5">
	<li>For the embedment of cells in the PuraMatrix prepare a 4-well cell culture plate and place a sterilised glass cover slip (diameter 13 mm) in two wells. Store the plate in the clean bench for the later use. Glass cover slips are only used for the immunocytochemical studies. If no immunocytochemical stainings are intended, the step can be skipped.</li>
</ol>
To prepare the hNPCs for encapsulation in 3D scaffolds one has to prepare the following solutions. Dilute the Benzonase in the Trypsin/EDTA solution, using a dilution factor of 1:10.000. For a Trypsin-Inhibitor/Benzonase solution mix: DMEM/F12 + Benzonase 25U/&mu;l + 1% HSA + Trypsin-inhibitor (0.5mg/ml).
<ol start="6">
	<li>Detach the cells by adding 500 &mu;l Trypsin/Benzonase solution and incubate for 5 min in an incubator (37&deg;C, 5% CO2). Stop the reaction with Trypsin-Inhibitor/Benzonase solution. Subsequently centrifuge the detached cells for 5 min at 3000 x g. Wash cells with 5 ml 10% Sucrose solution and centrifuge again. Discard the supernatant.</li>
	<li>Resuspend the cells in 10% sucrose solution, the final cell solution should contain 1x106 cells/ml.</li>
</ol>
The next steps, 1.8 to 1.11, should be done as fast as possible, because of the low pH of the PuraMatrix solution, which might be harmful for the cells.
<ol start="8">
	<li>Mix 60 &mu;l of cell suspension with solution 1.</li>
	<li>Add 120 &mu;l of solution 1, containing the cells, to the conical tube with solution 2 and mix carefully.</li>
	<li>Place 100 &mu;l of the mixture immediately on each cover slips resided in the 4-well plate.</li>
	<li>Add slowly 200 &mu;l cell culture medium per well and after 2-3 minutes another 200 &mu;l. This will initiate the self-assembly of the matrix.</li>
	<li>Incubate cells at 37 &deg;C, for 1h in the clean bench on a heating plate or the heated area of your clean bench. As far as possible do not move the plate as this might harm the assembling of the matrices.</li>
	<li>Remove most of the medium but not all, to avoid the matrices falling dry, with a 1000 &mu;l pipette. Add 500 &mu;l of medium to wash matrices for 10 min. Finally remove the medium and add 500 &mu;l fresh medium and place the culture plate in an incubator (37&deg;C, 5% CO2). As the matrices are sensitive to shock the plates should be moved as less as possible and stored, if possible, in a separate incubator.</li>
	<li>The here used hNPCs were proliferated for 7 days in the 3D-scaffolds and differentiation was initiated by withdrawal of the growth factors 13. Medium was changed every 2-3 days.</li>
</ol>
2. Part 2: Immunocytochemical staining of entire matrices
<ol>
	<li>In advance to the staining procedure one needs to prepare: a blocking buffer solution consisting of 5% normal goat serum and 0.3% of Triton X-100 dissolved in PBS, a 4% paraformaldehyde solution based on 0.1 M PBS and if needed a solution with 4',6-Diamidin-2'-phenylindoldihydrochlorid (DAPI) for a nuclei staining containing 100 ng DAPI/ml dissolved in PBS. Samples were mounted with a mixture of Mowiol and DABCO.</li>
	<li>To wash the matrices remove the medium with a 1000 &mu;l pipette and wash the scaffolds with PBS once for 5 minutes.</li>
	<li>To fix the scaffolds remove the PBS and add 400 &mu;l of paraformaldehyde (4% in 0.1 M PBS) to each well and incubate the matrices for 30 minutes at room temperature. Fixed Scaffolds can be stored in PBS supplemented with 0.02% NaN3 at 4&deg;C for several weeks up to several months.</li>
	<li>In advance of a staining wash the scaffolds with PBS for 5 minutes.</li>
	<li>Incubate the scaffolds in blocking buffer solution. Blocking solution was changed three times every 2-3 hours and subsequently scaffolds were incubated in blocking solution over night at 4&deg;C.</li>
	<li>Remove the blocking buffer solution and add the primary antibody dissolved in PBS supplemented with 1% normal goat serum and incubate the matrices over night at 4&deg;C.</li>
	<li>Remove the primary antibody solution and wash scaffolds 4 times for 2 h and subsequently over night at 4&deg;C with PBS.</li>
	<li>Remove the PBS and add the solution with the secondary antibody, dissolved in PBS supplemented with 1% normal goat serum, for 4 h at room temperature in the dark.</li>
	<li>Wash the samples with PBS 4-6 times for 1 h and subsequently over night at 4&deg;C in the dark.</li>
	<li>For a nuclei staining one can add 400 &mu;l of the DAPI solution for 30 min.</li>
	<li>To mount the samples one needs microscope slides. Add 50 &mu;l Mowiol/Dabco on the microscope slides. Remove the cover slips from the 4-well plate and put them carefully on the mounting medium.</li>
	<li>Here we used a Biozero 8000 microscope (Keyence, Germany, Karlsruhe) to obtain single micrographs and z-stacks. Each stack contains 30 single pictures with a distance of 1-2 &mu;m. Using the corresponding analyser software the blur inherent to fluorescence was removed before full projections of the z-stacks were produced.</li>
</ol>
3. Part 3: Release of hNPCs from the scaffolds for flow cytometry analysis
<ol>
	<li>In advance to the release of the cells from scaffolds wash the scaffolds with 500 &mu;l HBSS.</li>
	<li>Transfer the scaffolds by means of a 1000 &mu;l pipette to a 15 ml conical tube containing cell culture medium.</li>
	<li>To disrupt the scaffolds mechanically resuspend the cell solution several times with a 1000 &mu;l pipette and subsequently centrifuge the solution at 3000 x g for 5 min.</li>
	<li>Remove the supernatant with a pipette, add 500 &mu;l of Trypsin/Benzonase solution and resuspend the cell pellet several times.</li>
	<li>Incubate the cells for 5 min at 37&deg;C in the Trypsin/Benzonase solution. To stop the reaction add 1 ml Trypsin-inhibitor/Benzonase solution and pipette up and down several times.</li>
	<li>Centrifuge the cell suspension 5 min at 3000 x g, remove the supernatant and wash the cells with 2 ml HBSS buffer. Repeat this step two times. This procedure will remove matrix debris.</li>
	<li>Pass the cell suspension solution trough a cell strainer (pore size 70 &mu;m) to remove aggregates and collect the cells in cell culture medium. The obtained cells can be seeded again e.g. on cover slips for functional studies as patch clamp or fluometric measurements. To process the cells for flow cytometry, fix the cells immediately (see 4.1).</li>
</ol>
4. Part 4: Quantification of &beta;III-tubulin positive cells by flow cytometry
<ol>
	<li>Fix the released cells obtained in step 3.7 with 1% PFA for 15 min at room temperature.</li>
	<li>Centrifuge the cell suspension for 5 min at 3000 x g and remove the supernatant. If needed, the cells can be prepared to be stored at 4&deg;C for later analysis. Therefore resuspend cells in wash buffer (PBS supplemented with 0.5% BSA and 0.02% Na-azide).</li>
	<li>To proceed with the preparation for the flow cytometry, centrifuge the cell suspension for 10 min at 350 x g, discard the supernatant and resuspend the cells in 25 &mu;l of the antibody solution. Therefore the first antibody is mixed with saponin buffer at a concentration 1:100 e.g. &beta;III-tubulin. The saponin buffer consists of PBS with a saponin concentration of 0.5%, a BSA concentration of 0.5% and a Na-azide concentration of 0.02%.</li>
	<li>Incubate the cell suspension with the first antibody for 2 h at room temperature on a shaker. As a negative control prepare a sample without the first antibody.</li>
	<li>For the first washing step add 300 &mu;l saponin buffer directly to the cells. Centrifuge the cell suspension for 5 min at 350 x g. For the second washing step, discard the supernatant, add 300 &mu;l buffer and centrifuge the cell suspension for 5 min at 350 x g.</li>
	<li>Remove the supernatant and add 25 &mu;l solution containing the secondary antibody (e.g. Alexa Fluor 647, 1:1000, dissolved in saponin buffer). Incubate cells with the secondary antibody 1h at room temperature, in the dark.</li>
	<li>For the first washing step add 300 &mu;l saponin buffer directly to the cells. Centrifuge the cell suspension for 5 min at 350 x g. For the second washing step, discard the supernatant, add 300 &mu;l buffer and centrifuge the cell suspension for 5 min at 350 x g.</li>
	<li>Discard the supernatant and resuspend the cells in 500 &mu;l wash buffer for the flow cytometry analysis.</li>
</ol>
5. Representative Results
An example of hNPCs cultured in the self-assembling hydrogel scaffold PuraMatrix is shown in figure 1. The hNPCs grow in spheric like structures in unmodified PM. In this culture conditions, cells can hardly be recognised in a transmission light, although bundles of processes between the spheres are visible (fig 1A). Depending on the culture conditions of the used cell type, the matrix can be modified e.g. by adding laminin. For the hNPC cell line ReNcell VM (Millipore) laminin is necessary to induce a growth pattern with less dense aggregates but a more homogeneous distribution of the cells (fig 1B). Independent of modifications, the matrix can be used to study e.g. neuronal differentiation. Figure 1C presents a staining of the hNPCs for the neuronal marker &beta;III-tubulin. In this example the cells were grown for 7 days in the matrix and subsequently differentiated for 4 days, where differentiation was induced by withdrawal of the growth factors EGF and bFGF.13 Cell bodies and a dense network of processes built up by the cells can be recognised easily. However, it is obvious that a quantification of the cell number is time consuming, as a large number of pictures of different regions of the matrix has to be analysed, to obtain a reliable data base for a statistical analysis. In fig 1D one can see an example of cells released from the matrix and subsequently platted on cover slips. These cells might be used for functional studies.
The release of the cells from the 3D-scaffolds offers the possibility to analyse different parameters like expression of marker proteins or survival rate of the cells by AnnexinV/PI staining or a TUNEL assay. Figure 2 shows an example of a flow cytometry analysis of the percentage of &beta;III-tubulin+ cells. The negative control (cells not marked for &beta;III-tubulin) is shown in figure 2A. These controls are used to determine the gate for the later detection of &beta;III-tubulin+ cells (fig 2B). A comparison of a manual counting of cells and an analysis by flow cytometry is shown in figure 2C. The percentage of &beta;III-tubulin+ cells was determined by counting the total cell number (by means of a nuclear DAPI staining) and the number of &beta;III-tubulin+ cells in the fluorescence pictures.
<img alt="Figure 1" src="/files/ftp_upload/3830/3830fig1.jpg" /> 
Figure 1. hNPCs cultured in the self-assembling peptide hydrogel PuraMatrix. A) hNPCs encapsulated in PuraMatrix (PM) grow in spheric, dense packed structures, where the diameter of the spheres can be up to several hundred &mu;m. In between the spheres one can recognise bundles of processes built up by the cells (arrows). B) Cells encapsulated in PuraMatrix supplemented with laminin (PML) grow in less dense structures, more homogenously distributed. In the laminin supplemented matrix one can recognise single cells in less dense areas (arrows), however it is hardly possible to quantify the number of cells. C) hNPCs differentiated for 4 days in PML express neuronal marker like &beta;III-tubulin (green). To evaluate the percentage of positive cells one has to determine the total cell number by a nuclei staining like DAPI (blue). The microphotograph presents the full projection of a z-stack of pictures taken with Biozero-8000 microscope. D) Cells released from the matrix can be seeded on e.g. cover slips for further functional studies. The microphotograph shows cells cultured for 3d, subsequently to the release procedure. The staining for &beta;III-tubulin (red) reveals a comparable morphology to the cells hosted in the 3D scaffold.
<img alt="Figure 2" src="/files/ftp_upload/3830/3830fig2.jpg" /> 
Figure 2. Flow cytometry analysis cells released from PuraMatrix. To overcome the time consuming quantification of microphotographs we used a protocol to release cells cultured in PuraMatrix giving access to faster analysis by flow cytometry. A) Unstained cells were used as negative control, to set the gate (black frame) for the subsequently analysis of e.g. &beta;III-tubulin cells. B) To quantify the percentage of &beta;III-tubulin+ cells the same gate, set in the negative control, was used. Positive cells appear in the right part of the x-axis, where also an intermediate population was observed (dotted frame), most likely representing cell debris. C) The comparison of manual counted cells and cells counted by flow cytometry revealed a much higher proportion of positive cells, where the time dependency of the number of &beta;III-tubulin+ was comparable, indicating the reliability of the flow cytometry protocol.

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No conflicts of interest declared.

The use of 3D-scaffolds offers the opportunity to study the development of different cell types in a cell culture situation closer to the in vivo situation. However, regarding the analysis of e.g. neuronal differentiation or functional studies one has to overcome some obstacles to gain reliable data for e.g. quantification of cell types.
Here we described the culture of hNPCs in the peptide hydrogel based scaffold PuraMatrix and the subsequently release of the cells to be used for the...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>PuraMatrix peptide hydrogel </td>
 <td>BD Bioscience </td>
 <td>354250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse laminin I</td>
 <td>Cultrex</td>
 <td>400-2009090 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma</td>
 <td>S9378-1KG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Normal goat serum</td>
 <td>Dako</td>
 <td>X0907</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Triton X 100</td>
 <td>Roth</td>
 <td>3051.3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS Dulbecco</td>
 <td>Biochrom AG</td>
 <td>L 1825</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Gibco</td>
 <td>14170-088</td>
 <td>Hanks' Balanced Salt Solution 1X</td>
 </tr>
 <tr>
 <td>&beta;III-tubulin antibody</td>
 <td>Santa Cruz</td>
 <td>Sc-51670</td>
 <td>Mouse, monoclonal, 1:500</td>
 </tr>
 <tr>
 <td>Alexa Fluor 488</td>
 <td>Invitrogen</td>
 <td>A 11029</td>
 <td>Goat &alpha; mouse, 1:1000</td>
 </tr>
 <tr>
 <td>Alexa Fluor 568</td>
 <td>Invitrogen</td>
 <td>A 11031</td>
 <td>Goat &alpha; mouse, 1:1000</td>
 </tr>
 <tr>
 <td>Alexa Fluor 647</td>
 <td>Invitrogen</td>
 <td>A 21235</td>
 <td>Goat &alpha; mouse, 1:1000</td>
 </tr>
 <tr>
 <td>Mowiol 4-88 Reagent</td>
 <td>Calbiochem</td>
 <td>475904</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dabco</td>
 <td>Aldrich</td>
 <td>D2,780-2</td>
 <td>1,4-Diazabicyclo[2.2.2]octane 98%</td>
 </tr>
 <tr>
 <td>Cell strainer</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td>70 &mu;m pore size</td>
 </tr>
 <tr>
 <td>Saponin</td>
 <td>Merck</td>
 <td>7695</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin/ EDTA</td>
 <td>GIBCO</td>
 <td>25300-054</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Benzonase 250 U/&mu;l</td>
 <td>Merck</td>
 <td>1.01654.0001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin Inhibitor</td>
 <td>Sigma</td>
 <td>T6522 (500 mg)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>20% HSA</td>
 <td>Octapharma</td>
 <td>Human-Albumin Kabi 20%</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel at JoVE.com

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

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 ...

cultivation-human-neural-progenitor-cells-3-dimensional-self

Research

JoVE Journal

Bioengineering

16.5K 조회수.  University of Rostock. 여기서 우리는 자기 조립 3 차원 비계 문화에 인간 신경 전구 세포의 사용을 설명합니다. 우리는 유동세포계측법에 의해 연속적 예 분석하는 공사장 공중 발판에서 세포를 릴리스하는 프로토콜을 제시한다. 이 프로토콜은 상세한 mechanistically 연구를 수행하는 다른 세포 유형에 맞게 수 있습니다.

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

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

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 ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...