Name: A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids (Video) | JoVE Journal
Uploaded: 2011-05-06T16:00:00.000+00:00
Duration: 7 min 24 s
Description: Learn a rapid hanging drop method to generate 3D tissue-like spheroids for studying cell-cell interactions in cell biology and tissue engineering research. This technique forms aggregates in 24-48 hours.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The author would like to thank Dr. Dongxuan Jia for technical assistance. Some of the images appearing in this article were in collaboration with Dr. Malcolm S. Steinberg, Department of Molecular Biology, Princeton University. The author would also like to thank the Department of Defense Prostate Cancer Research Program (grants PC-030482 and PC-991552) and the NCI/NIH (grant R01CA118755)for their generous support.

1. Preparation of a Single Cell Suspension
<ol>
	<li>Adherent cell cultures should be grown to 90% confluence, whereupon monolayers should be rinsed twice with PBS. After draining well, add 2 mls (for 100 mm plates) of 0.05% trypsin-1 mM EDTA, and incubate at 37°C until cells detach. Stop trypsinization by adding 2 mls of complete medium and gently use a 5 ml pipette to triturate the mixture until cells are in suspension. Transfer cells to a 15 ml conical tube.</li>
	<li>Add 40 μl of a 10 mg/ml DNAse stock and incubate for 5 minutes at RT. Vortex briefly and centrifuge at 200 XG for 5 minutes.</li>
	<li>Discard supernatant and wash pellet with 1 ml complete tissue culture medium. Repeat, then resuspend cells in 2 mls of complete tissue culture medium.</li>
	<li>Count the cells using a hemacytometer, or automated cell counter and adjust concentration to 2.5 x 106 cells/ml. For this demonstration a BioRad TC10 automated cell counter was used.</li>
</ol>
2. Formation of Hanging Drops.
<ol>
	<li>Remove the lid from a 60 mm tissue culture dish and place 5 mls of PBS in the bottom of the dish. This will act as a hydration chamber.</li>
	<li>Invert the lid and use a 20 μl pipettor to deposit 10 μl drops onto the bottom of the lid. Make sure that drops are placed sufficiently apart so as to not touch. It is possible to place at least 20 drops per dish.</li>
	<li>Invert the lid onto the PBS-filled bottom chamber and incubate at 37°C/5% CO2/95% humidity, monitor the drops daily and incubate until either cell sheets or aggregates have formed. A stereo microscope can be used to assess aggregate formation.</li>
	<li>Once sheets form, they can be transferred to round-bottom glass shaker flasks containing 3 mls of complete medium and incubated in a shaking water bath at 37°C and 5% CO2 until spheroids form.</li>
</ol>
3. Notes
<ol>
	<li>Depending on cell size, concentration may need to be adjusted up- or down.</li>
	<li>Cells can be stained with membrane intercalating fluorescent dyes prior to hanging drop formation.</li>
	<li>Two different cell types can be differentially stained and mixed in a 1:1 (or other) ratio prior to formation of hanging drops. These can be cancer and stromal cells, embryonic tissues, or cells genetically engineered to have specific adhesive signatures.</li>
	<li>Cells can be detached from tissue culture dishes using 0.05% trypsin/2 mM calcium to preserve cadherin function.</li>
	<li>Depending on the experiment, sheet sizes can either be measured, or aggregates can be used for mechanical testing or for biochemical or molecular analysis.</li>
</ol>
4. Representative Results:
Sheet or spheroid formation typically occurs within 24 hours but may take longer depending on cell type. Figure 1 represents images captured after 18 hours of incubation of untreated prostate cancer MAT-LyLu (MLL) cells in hanging drop culture (Figure 1A) or cells treated with 25 μm MEK inhibitor PD98059. Note that MEKi treatment results in a marked compaction of the cell sheet. Compaction can be quantified by measuring the size of 10-20 (or more) hanging drops and comparing average size between treatment groups. We typically analyze images using ImageJ software by thresholding each image then converting images to Binary Mode, whereupon we apply particle analysis to reveal the total image area in pixels. This can then be easily converted to square microns. Figure 2 represents a size comparison for untreated and MEKi-treated MLL cells. As noted, MEKi treatment significantly reduces sheet size as compared by Student's t-test (P&lt;0.0001). For this comparison, 10 aggregates for each of untreated and treated cells were measured. Figure 3 represents a chick embryonic liver spheroid formed after 24 hours in hanging drop culture and 2 days in a shaker bath.
The hanging drop assay can also be modified to include more than one cell type to reveal the spatial positioning adopted. For example, chick embryonic liver cells can be isolated, stained with a fluorescent membrane-intercalating dye and mixed with chick embryonic heart cells that have been stained with a different fluorescent marker. Figure 4A shows that rather than remaining intermixed, liver and heart cells tend to "sort-out" from one-another and adopt a sphere-within-a-sphere configuration in which heart cells are enveloped by liver cells. This configuration can be explained by differences in intercellular adhesion between liver and heart cells. This concept is codified in the Differential Adhesion Hypothesis which has been used to explain cell sorting behavior between embryonic germ layer tissue for amphibian3and teleost 4embryos, for pancreatic islet cell organization 5and for cell sorting between two cell populations identical in every respect other than for the expression levels of the same type of cadherin (6and Figure 4B). This result is particularly important for demonstrating how engineering a simple difference in adhesion between cell populations may result in the generation of organ structure, for example, a pancreatic islet 5.
 <img src="/files/ftp_upload/2720/2720fig1.jpg" alt="Cell aggregation comparison, microscopy image; A: large aggregate, B: small aggregate for research." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Images of cells incubated in hanging drop culture for 18 hours either in the absence (A) or presence (B) of 25 μm of the MEK inhibitor PD98059. Images were captured using a Nikon Eclipse TE 300 epifluorescence microscope and a Photometrics CoolSnap ES digital camera.
 <img src="/files/ftp_upload/2720/2720fig2.jpg" alt="Bar chart comparing aggregate size of MLL and MLL+MEKi for statistical analysis." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Quantification of MEKi-induced compaction of rat prostate cancer MLL cells. Hanging drop cultures of untreated and MEKi-treated MLL cells were incubated for 18 hours whereupon aggregate size was determined as described above. Statistical analysis of aggregate size was by Student's t-test. The asterisk represents a statistically significant difference in aggregate size (P&lt;0.0001) between untreated and MEKi-treated cells and suggests that MEKi-treatment results in significantly smaller and more compact aggregates.
 <img src="/files/ftp_upload/2720/2720fig3.jpg" alt="Static equilibrium; sphere on edge; physics diagram; stability; balance analysis." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. A spheroid formed by incubating chick embryonic liver cells in hanging drop culture for 18 hours then in an incubator/shaking bath for 48 hours. This image was captured using a Nikon SMZ800 stereo microscope and a Sony Black CCD camera.
 <img src="/files/ftp_upload/2720/2720fig4.jpg" alt="Fluorescent microscopy image, cellular structure, emission analysis, color-coded results." data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4. Hanging drop culture reveals sorting-out behavior between different cell types. In panel A, chick embryonic liver cells were stained with PKH-2 membrane intercalating dye and mixed in equal proportions with PKH-26 stained chick embryonic heart cells. In panel B, two N-cadherin-transfected L cell clones (LN4 and LN2a), expressing N-cad at their surfaces in the ratio of 2.4:1, were also stained with the fluorescent membrane intercalating dyes PKH-2 and PKH-26, (Sigma-Aldrich), mixed in equal proportions and cultured as hanging drops7. After 18 hours in culture, cell sheets were transferred to shaking flasks and incubated for another 48 hours. Aggregates were fixed in 2% paraformaldehyde in phosphate-buffered saline and viewed with a BioRad MRC600 scanning laser confocal microscope system attached to a Nikon Optiphot-2 microscope. Optical sections from both green and red channels were collected and a Silicon Graphix Crimson VGX workstation equipped with Vital Images Voxel View Ultra imaging software was used to assign false color to both channels and to merge the images, revealing the anatomical configurations generated.(A) Confocal optical section through an aggregate demonstrating envelopment of heart cells (here in a pseudo-color yellow) by liver cells in pseudocolor blue (from 8). (B) Confocal optical section through an aggregate demonstrating envelopment of high N-cadherin expressing cells (green) by those expressing lower levels of N-cadherin (red) (from 6).

<ol><li>Cukierman, E., Pankov, R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+interactions+with+three-dimensional+matrices%5BTitle%5D)+AND+%22Curr+Opin+Cell+Biol%22%5BJournal%5D)">Cell interactions with three-dimensional matrices.</a> Curr Opin Cell Biol. 14, 633-639 (2002).</li>
<li>Nelson, C. M., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Of+extracellular+matrix%2C+scaffolds%2C+and+signaling%3A+tissue+architecture+regulates+development%2C+homeostasis%2C+and+cancer%5BTitle%5D)+AND+%22Annu+Rev+Cell+Dev+Biol%22%5BJournal%5D)">Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer.</a> Annu Rev Cell Dev Biol. 22, 287-309 (2006).</li>
<li>Davis, G. S., Phillips, H. M., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germ-layer+surface+tensions+and+%26quot%3Btissue+affinities%26quot%3B+in+Rana+pipiens+gastrulae%3A+quantitative+measurements%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Germ-layer surface tensions and &quot;tissue affinities&quot; in Rana pipiens gastrulae: quantitative measurements.</a> Dev Biol. 192, 630-644 (1997).</li>
<li>Schotz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+differences+in+tissue+surface+tension+influence+zebrafish+germ+layer+positioning%5BTitle%5D)+AND+%22HFSP+J%22%5BJournal%5D)">Quantitative differences in tissue surface tension influence zebrafish germ layer positioning.</a> HFSP J. 2, 42-56 (2008).</li>
<li>Jia, D., Dajusta, D., Foty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+surface+tensions+guide+in+vitro+self-assembly+of+rodent+pancreatic+islet+cells%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Tissue surface tensions guide in vitro self-assembly of rodent pancreatic islet cells.</a> Dev Dyn. 236, 2039-2049 (2007).</li>
<li>Foty, R. A., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+differential+adhesion+hypothesis%3A+a+direct+evaluation%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">The differential adhesion hypothesis: a direct evaluation.</a> Dev Biol. 278, 255-263 (2005).</li>
<li>Li, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+the+casein+kinase+2+subunits+in+Chinese+hamster+ovary+and+3T3+L1+cells+provides+information+on+the+role+of+the+enzyme+in+cell+proliferation+and+the+cell+cycle%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Expression of the casein kinase 2 subunits in Chinese hamster ovary and 3T3 L1 cells provides information on the role of the enzyme in cell proliferation and the cell cycle.</a> J Biol Chem. 274, 32988-32996 (1999).</li>
<li>Foty, R. A., Pfleger, C. M., Forgacs, G., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+tensions+of+embryonic+tissues+predict+their+mutual+envelopment+behavior%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Surface tensions of embryonic tissues predict their mutual envelopment behavior.</a> Development. 122, 1611-1620 (1996).</li>
<li>Wendt, D., Riboldi, S. A., Cioffi, M., Martin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+and+bottlenecks+of+bioreactors+in+3D+cell+culture+and+tissue+manufacturing%5BTitle%5D)+AND+%22Adv+Mater%22%5BJournal%5D)">Potential and bottlenecks of bioreactors in 3D cell culture and tissue manufacturing.</a> Adv Mater. 21, 3352-3367 (2009).</li>
<li>Berry, D. P., Harding, K. G., Stanton, M. R., Jasani, B., Ehrlich, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+wound+contraction%3A+collagen+organization%2C+fibroblasts%2C+and+myofibroblasts%5BTitle%5D)+AND+%22Plast+Reconstr+Surg%22%5BJournal%5D)">Human wound contraction: collagen organization, fibroblasts, and myofibroblasts.</a> Plast Reconstr Surg. 102, 124-131 (1998).</li>
<li>Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblast+biology+in+three-dimensional+collagen+matrices%5BTitle%5D)+AND+%22Trends+Cell+Biol%22%5BJournal%5D)">Fibroblast biology in three-dimensional collagen matrices.</a> Trends Cell Biol. 13, 264-269 (2003).</li>
<li>Wang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reciprocal+interactions+between+beta1-integrin+and+epidermal+growth+factor+receptor+in+three-dimensional+basement+membrane+breast+cultures%3A+a+different+perspective+in+epithelial+biology%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology.</a> Proc Natl Acad Sci U S A. 95, 14821-14826 (1998).</li>
<li>Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversion+of+the+malignant+phenotype+of+human+breast+cells+in+three-dimensional+culture+and+in+vivo+by+integrin+blocking+antibodies%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.</a> J Cell Biol. 137, 231-245 (1997).</li>
<li>Boudreau, N., Werb, Z., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppression+of+apoptosis+by+basement+membrane+requires+three-+dimensional+tissue+organization+and+withdrawal+from+the+cell+cycle%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Suppression of apoptosis by basement membrane requires three- dimensional tissue organization and withdrawal from the cell cycle.</a> Proc Natl Acad Sci U S A. 93, 3509-3513 (1996).</li>
<li>Croix, B., Rak, J. W., Kapitain, S., Sheehan, C., Graham, C. H., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversal+by+hyaluronidase+of+adhesion-dependent+multicellular+drug+resistance+in+mammary+carcinoma+cells%5BTitle%5D)+AND+%22J.+Nat.+Cancer+Inst%22%5BJournal%5D)">Reversal by hyaluronidase of adhesion-dependent multicellular drug resistance in mammary carcinoma cells.</a> J. Nat. Cancer Inst. 88, 1285-1296 (1996).</li>
<li>Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+structure%2C+nuclear+organization%2C+and+gene+expression+in+normal+and+malignant+breast%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Tissue structure, nuclear organization, and gene expression in normal and malignant breast.</a> Cancer Res. 59, 1757-1763 (1999).</li>
<li>Marrero, B., Messina, J. L., Heller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+a+tumor+spheroid+in+a+microgravity+environment+as+a+3D+model+of+melanoma%5BTitle%5D)+AND+%22In+Vitro+Cell+Dev+Biol+Anim%22%5BJournal%5D)">Generation of a tumor spheroid in a microgravity environment as a 3D model of melanoma.</a> In Vitro Cell Dev Biol Anim. 45, 523-534 (2009).</li>
<li>Napolitano, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scaffold-free+three-dimensional+cell+culture+utilizing+micromolded+nonadhesive+hydrogels%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels.</a> Biotechniques. 43, 494-496 (2007).</li>
<li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrogels+as+extracellular+matrix+mimics+for+3D+cell+culture%5BTitle%5D)+AND+%22Biotechnol+Bioeng%22%5BJournal%5D)">Hydrogels as extracellular matrix mimics for 3D cell culture.</a> Biotechnol Bioeng. 103, 655-663 (2009).</li>
</ol>

No conflicts of interest declared.

Studies have shown that culturing cells in a three-dimensional context (3D) produces distinct cellular morphology and signaling when compared to a rigid two-dimensional (2D) culture system9. For example, fibroblast-populated collagen gels demonstrate that fibroblast morphology in 3D is quite distinct from that observed in 2D 10,11. Similarly, 3D culture can induce tissue-specific differentiation of mammary epithelial cells. 3D culture systems have been used to distinguish between normal and malignan...

<table><tbody><tr><td>automatic cell counter</td><td>Bio-Rad</td><td>TC10</td><td></td></tr><tr><td>shaking water bath with CO&lt;sub&gt;2&lt;/sub&gt; gable cover</td><td>Labline Instruments</td><td>3540</td><td></td></tr></tbody></table>

a simple hanging drop cell culture protocol for generation of 3d spheroids

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 ...

The aim of this experiment is to generate 3D tissue like steroids. This is accomplished by first harvesting cultured cells and then generating a single cell suspension. Next drops of the cell suspension, a deposited onto the underside of the inverted lid from a tissue culture dish.Then the lid is inverted onto the hydration chamber. The final step is to incubate the hanging drops for 24 to 48 hours to allow aggregates to form. These can then be transferred to a shaking water bath and incubated for up to five more days.Ultimately, results can be obtained that show differences in aggregate cohesion or cell sorting through compaction assays tissue surface 10 symmetry or epi fluorescence microscopy. The main advantage of this technique over existing methods like conventional 2D tissue culture, for example, is that cells grown in 3D more closely mimic cellular interactions experienced by cells in a tissue environment. I'll be demonstrating this method to show you that it's so simple that even a PI can do it.To begin this experiment, harvest adherence cells at 90%co fluency. First, aspirate the culture media and rinse the cell sheet twice with PBS. Then aspirate the PBS and pipette two milliliters of 0.05%trips in EDTA solution onto the monolayer layer.Place the plate into the 37 degree Celsius tissue culture incubator and incubate until cells detach. Next, remove the plate from the incubator and halt ization. Adding two milliliters of complete medium.Use a five milliliter pipette to tritrate the mixture until the cells are fully in suspension. Then transfer the cell suspension into a 15 milliliter conical tube. Pipette 40 microliters of a 10 milligrams per milliliter, DNA solution into the cell suspension, and incubate it room temperature for five minutes following incubation.Vortex the cell suspension briefly and then centrifuge at 200 times gravity for five minutes. Once the centrifugation is complete, aspirate the S supernatant and discard, then resuspend the palette in one milliliter of complete medium to wash. Repeat the centrifugation and Reese from the pellet in one milliliter of complete medium.Next, perform a cell count using an automated cell counter or hemo cytometer. Adjust the cell suspension with media to a final concentration of 2.5 times 10 to the six cells per milliliter. Then proceed to the formation of hanging drops.Prepare a hydration chamber by removing the lid from a 60 millimeter tissue culture dish and pipetting five milliliters of PBS into the bottom of the dish. Invert the lid of the plate and pipette up to 2010 microliter drops of cell suspension onto the underside of the lid. Ensure that the drops are placed sufficiently apart so as not to touch.Gently replace the lid containing the drop cultures back onto the bottom of the PBS containing dish. Place the culture plate into a tissue culture incubator. Set at 37 degrees Celsius, 5%carbon dioxide and 95%humidity and incubate overnight.Monitor the drops daily and continue the incubation until either cell sheets or aggregates have formed. Once cell sheets or aggregates have formed, transfer them into round bottom glass shaker flasks containing three milliliters of complete medium. Incubate the flasks containing the cell sheets or aggregates in a shaking water bath.Maintained at 37 degrees Celsius and 5%carbon dioxide for around 24 hours until the formation of steroid can be visualized under a dissecting microscope. Once steroids have been formed, the effect of experimental manipulations on steroid formation may be quantified according to the procedure outlined in the written protocol. Also, the membrane intercalation of any previously included fluorescent dyes may now be visualized using fluorescence microscopy.This brightfield microscope image demonstrates the aggregation of rat prostate cancer MLL cells after 18 hours of incubation using the hanging drop method. This image shows a second aggregate of MLL cells formed after 18 hours of culture using the hanging drop method. These cells were cultured in the presence of 25 micromolar of the MEK inhibitor PD 9 8 0 5 9 resulting in a substantially more compact aggregate of cells.Here, the difference between MEK inhibitor treated MLL hanging drop culture, aggregate size and untreated control aggregate size is demonstrated. The asterisk represents a statistically significant difference of P less than 0.0001. As determined by students T-test between untreated and MEK inhibitor treated aggregates and suggests that MEK inhibitor treatment results in significantly smaller and more compact aggregates.This image was captured using a Nikon Smz 800 stereo microscope and illustrates a steroid formed by incubating chick embryonic liver cells in hanging drop culture for 18 hours. Then in a 37 degrees Celsius shaking water bath for 48 hours. This confocal microscope image demonstrates the sorting out behavior that occurs when certain cell populations are co cultured using the hanging drop method before proceeding to hanging drop culture chick embryonic liver cells were stained with pkh two membrane inter coating dye and chick embryonic.Heart cells were stained with PKH 26. The cell suspensions were then mixed and hanging drop. Culture was performed for 18 hours followed by 48 hours of incubation in the 37 degrees Celsius shaking water bath.It can be seen from the optical section that the heart cells colored yellow are enveloped by liver cells. Pseudo colored blue here, two nardi and transfected L cell clones expressing NCA at their surfaces. At a ratio of 2.4 to one, were stained with the fluorescent membrane into collating dyes, PKH two and PK KH 26 mixed in equal proportions and cultured as hanging drops as before.In this example, the high-end cadian expressing cells, which are pseudo colored green are enveloped by those expressing lower levels of en cadian, which are pseudo colored red. Once mastered, this technique can be done in 30 minutes or less if it's performed properly.

a-simple-hanging-drop-cell-culture-protocol-for-generation-3d

Research

JoVE Journal

Bioengineering

Spheroids are a type of 3D cell culture model used to more accurately represent a cell’s environment compared to 2D cell culture monolayer models. In the example protocol, we will see scientists generate spheroids from a colorectal cancer cell line and apply live-cell imaging to track the spheroids’ growth.

Spheroid Generation from Cell Lines: A Three-Dimensional (3D) Cell Culture Method

This video describes the technique of generating three-dimensional spheroids via the hanging drop method using melanoma cells. The generated in vitro three-dimensional (3D) organotypic melanoma spheroid model can portray the in vivo architecture of malignant melanoma and may give insights into intra-tumoral...

Hanging Drop Method: A Technique to Generate 3D Melanoma Spheroids

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix.  Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen.  Invasion out of the spheroids is subsequently monitored.

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates’ potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Learn a rapid hanging drop method to generate 3D tissue-like spheroids for studying cell-cell interactions in cell biology and tissue engineering research. This technique forms aggregates in 24-48 hours.

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

Explore More Videos