Hanging Drop System Research Articles

Modeling Tumor Development, Progression and Metastasis in 3D Artificial Tissues: Characterization of Structure and Labeling Patterns in Breast Cancer Hanging‐drop Spheroid Cultures

In this report our laboratory presents the results of a series of studies being conducted to explore the application of 3D artificial tumor tissues as in‐vitro models of breast cancer development, progression and metastasis. In this case, we have employed MCF‐7 breast ductal adenocarcinoma cells and 3D Biomatrix hanging drop plates to evaluate these tumor properties within the context of staged tumor spheroids. A consistent observation of ours in nearly a decade of modeling breast cancer using various natural 3D scaffolds and hydrogel matrices has been that solid tumor models grown in long‐term culture predictably begin to shed both single cells and cell clusters in the form of small groupings of cells as well as progressively larger spheroids. Although we have shown that at least some of these structures have properties consistent with metastasis, including attachment and invasion of target tissues, spontaneously generated versions of cancer clusters also present as pleiomorphic structures and in relatively small numbers which complicates their study. Therefore, we sought to use a system that generates identical clusters and spheres from specific numbers of suspended single cells. The hanging drop system we employ comes in a 96 well format that allows us to generate literally as many spheres as we need for each study. Furthermore, these spheres can be grown out to match any of the sizes commonly found in our tumor models thereby allowing us to study each stage for structure, cell populations and markers. In addition these spheres are also assisting us in systematically studying the process of cancer cell cluster attachment and invasion and to test our working hypothesis that this is controlled through activation of EMT‐like responses through CD44 binding of hyaluronate on the target tissue. Using direct labeling of intact spheres coupled to embedding and sectioning followed by immunolabeling, these studies are demonstrating that over a period of 144 hours our spheroid cultures progress from simple clusters of a few cells to aggregated spheres containing thousands of cells and measuring up to ~600um in diameter. By this later stage, many spheres actually begin to generate their own smaller spheres and clusters as well. In our view this reflects the concept that past 48 hours in culture these spheres transition into being actual tumor organoids themselves. This is supported by development of a complex CD24+ internal structure distinct from an outer shell of CD44+ cells. These studies are ongoing and support our view that 3D artificial tumors are effective tools to study cancer biology in‐vitro.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Surface-Engineered Paper Hanging Drop Chip for 3D Spheroid Culture and Analysis.

Protein corona coated onto the hydrophilic cellulose fiber turns into hydrophobic upon UV irradiation without hindering the porosity of the paper while simultaneously reducing nonspecific adsorption. Taking advantage of the biofouling-resistant, hydrophobic, and fluid transport through property, we demonstrated hanging drop three-dimensional (3D) spheroid culture and in-site analysis, including drug testing, time-dependent detection of secreted protein, and fluorescence staining without disturbing the spheroids. This single hanging drop system can also be extended to a networked hanging drop chip to mimic in vivo microphysiology by combining with wax-patterned microfluidic channels, where well-to-well interaction can be accurately controlled in a passive manner. As a proof of concept, the effects of a concentration gradient of nutrient and variable dosage of anticancer drugs were studied in the 3D spheroids cultured on paper. The experimental results suggested that a complex network device could be fabricated on a large scale on demand at a minimal cost for 3D spheroid culture. Our method demonstrates a future possibility for paper as a low cost, high-throughput 3D spheroid-based "body-on-a-chip" platform material.

Advanced Biosystems: Looking Back on our First Year and Welcome to 2018

<i>Advanced Biosystems</i>: Looking Back on our First Year and Welcome to 2018

Coculture of Spheroids/2D Cell Layers Using a Miniaturized Patterned Platform as a Versatile Method to Produce Scaffold‐Free Tissue Engineering Building Blocks

AbstractA hanging drop system that allows the multiconfigurational coculture of 3D microtissues is suggested as a versatile platform to promote the formation of functional preconditioned spheroids/microtissues, namely stem‐cell‐derived tissue engineering microtissues, with potential to be applied as scaffold‐free building blocks. Here, superhydrophobic (SH) platforms patterned with wettable regions are adapted for the production and culture of human adipose‐derived stem‐cell spheroids under indirect coculture with 2D layers of different cell types and direct coculture setups. The versatile indirect and direct coculture setups allow the use of cell lines as soluble biomolecules “factories” to continuously modulate microtissues response aspects, including their viability, cell number, and protein expression. This novel application of patterned SH platforms may find application in the massive production of modulated spheroids for the biomedical and pharmaceutical fields, with specific added value in the biofabrication of 3D constructs for tissue regeneration, as disease models, or even for organoids preparation.

The influence of stromal cells and tumor-microenvironment-derived cytokines and chemokines on CD3+CD8+ tumor infiltrating lymphocyte subpopulations

ABSTRACT The tumor microenvironment has been identified as a major mediator of immunological processes in solid tumors. In particular, tumor-associated fibroblasts are known to interact with tumor infiltrating immune cells. We describe the influence of fibroblasts and tumor-microenvironment-derived cytokines on the infiltration capacity of CD3+CD8+ cytotoxic T lymphocyte subpopulations using a multicellular 3D co-culture system. 3D tumor microtissues were cultivated using a hanging drop system. Human A549 and Calu-6 cancer cell lines were incubated alone or together with the human fibroblast cell line SV80 for 10 d to form microtissues. On day 10, peripheral blood mononuclear cells (PBMC) were added with or without cytokine stimulation for 24 h. Infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of the microtissues and the infiltration of the PBMCs were analyzed by immunohistochemistry, and endogenous cytokine and chemokine expression was analyzed with a multi-cytokine immunoassay. Secretion of chemokines is increased in microtissues consisting of cancer cells and fibroblasts. PBMC infiltrate the whole spheroid in cancer cell monocultures, whereas in co-cultures of cancer cells and fibroblasts, PBMCs are rather localized at the margin. Activated CD69+ and CD49d+ T lymphocytes show an increased microtissue infiltration in the presence of fibroblasts. We demonstrate that the stromal component of cancer microtissues significantly influences immune cell infiltration. The presence of fibroblasts in cancer microtissues induces a shift of T lymphocyte infiltration toward activated T lymphocytes.

High Quality Multicellular Tumor Spheroid Induction Platform Based on Anisotropic Magnetic Hydrogel.

In recent years, multicellular spheroid (MCS) culture has been extensively studied both in fundamental research and application fields since it inherits much more characteristics from in vivo solid tumor than conventional two-dimensional (2D) cell culture. However, anticell adhesive MCS culture systems such as hanging drop allow certain cell lines only to form loose, irregular aggregates rather than MCS with physiological barriers and pathophysiological gradients, which failed to mimic in vivo solid tumor in these aspects. To address this issue, we improved our previously established anisotropic magnetic hydrogel platform, enabling it to generate multicellular spheroids with higher efficiency. The qualities of multicellular tumor spheroids (MCTSs) obtained on our platform and from classic 3D culture systems were compared in terms of morphology, biological molecule expression profiles, and drug resistance. In this novel platform, mature MCTSs with necrotic cores could be observed in 1 week. And results of molecular biological assays with real time-PCR and western-blot confirmed that MCTSs obtained from our platform performed higher cell pluripotency than those obtained from the hanging drop system. Moreover, a lower cell apoptosis ratio and better viability of cancer cells were observed on our platform both under culturing and drug treatment. In conclusion, higher quality of MCTSs obtained from this anisotropic magnetic hydrogel than classic hanging drop system validate its potential to be an in vitro platform of inducing tumor MCTS formation and drug efficacy evaluation.

3D Hanging Drop Culture to Establish Prostate Cancer Organoids.

Three-dimensional (3D) cell culture enables the growth of cells in a multidimensional and multicellular manner compared to conventional cell culture techniques. Especially in prostate cancer research there is a big need for more tissue-recapitulating models to get a better understanding of the mechanisms driving prostate cancer as well as to screen for more efficient drugs that can be used for treatment. In this chapter we describe a 3D hanging drop system that can be used to culture prostate cancer organoids as tumor epithelial monocultures and as epithelial-stromal cocultures.

Sliced Magnetic Polyacrylamide Hydrogel with Cell-Adhesive Microarray Interface: A Novel Multicellular Spheroid Culturing Platform.

Cell-adhesive properties are of great significance to materials serving as extracellular matrix mimics. Appropriate cell-adhesive property of material interface can balance the cell-matrix interaction and cell-cell interaction and can promote cells to form 3D structures. Herein, a novel magnetic polyacrylamide (PAM) hydrogel fabricated via combining magnetostatic field induced magnetic nanoparticles assembly and hydrogel gelation was applied as a multicellular spheroids culturing platform. When cultured on the cell-adhesive microarray interface of sliced magnetic hydrogel, normal and tumor cells from different cell lines could rapidly form multicellular spheroids spontaneously. Furthermore, cells which could only form loose cell aggregates in a classic 3D cell culture model (such as hanging drop system) were able to be promoted to form multicellular spheroids on this platform. In the light of its simplicity in fabricating as well as its effectiveness in promoting formation of multicellular spheroids which was considered as a prevailing tool in the study of the microenvironmental regulation of tumor cell physiology and therapeutic problems, this composite material holds promise in anticancer drugs or hyperthermia therapy evaluation in vitro in the future.

Abstract 4672: Development of 2D - cell strain and 3D - tumor spheroid models for Precision Medicine

Abstract Background Precision oncology is a clinical approach aimed towards tailoring treatment strategies for patients based on the genetic profile of each patient's malignancy. Available cell line models are greatly limited in recapitulating the genetic profile of the tumor and tumor microenvironment of patients and therefore limit preclinical evaluation of new targeted agents. Furthermore, a high failure rate of drug candidates can be attributed in part to use of two dimensional (2D) monolayer cultures as the initial screening method that frequently produces inaccurate results and does not predict chemoresistance. The ability of three dimensional (3D) cultured tumor cells derived directly from patients to recapitulate tumor-like microenvironment suggests that engineered tumor models may provide more accurate and reproducible information for drug testing. At the Institute for Precision Medicine, we grow patient-derived 2D cell strains and engineer 3D tumor spheroids for patient`s tumor specific drug testing. Design Fresh tissue samples from 30 different solid tumors were digested in Type II or IV Collagenase (Gibco) and then re-suspended in growth factor reduced Matrigel (BD), plated on plastic, and overlaid with cell type specific cell growth media tsCM, which consists of either serum free Advanced DMEM/F12 (Gibco) with multiple growth factors optimized to propagate benign primary tumor cells, or FBS enriched DMEM/F12 media. Cultures were maintained at 37°C in 5% CO2. Once established, early cell strain culture passage and tumor spheroids size &amp;lt; 500 μm were cryopreserved. Specific drug testing was performed following 3 models: 2D monolayer culture, 2D monolayer culture converted in a 3D system using the hanging drop system from Biomatrix 3D®, and engineered 3D tumor spheroids. Results Our success rate in generating 2D- cell strains and 3D tumor spheroids is about 30%-40%. In the initial 30 samples, we were able to establish 11 continuous 2D and 3D tumor spheroid cultures derived from distinct primary and metastatic cancers including prostate, kidney, bladder and endometrium, as well as sarcomas and a neuroendocrine tumor. Tumor content in biopsy represents a major factor for successful growth. Histology and cytology examination demonstrates that 3D tumor spheroids recapitulate harvested tumors. Next generation sequencing shows the presence of same genomic alterations compared with original tumor without generating additional mutations. Conclusion We have developed individual patient-derived 2D tumor cell strains and 3D engineered tumor spheroids as well as models for patient`s tumor specific drug testing. This effort has been implemented for Precision Care and Clinical Trials at our Institution. Citation Format: Chantal Pauli, Jonathan Pauwels, Myriam Kossai, Nikolai Steklov, Andrea Sboner, Rema Rao, Kenneth Hennrick, Brian Robinson, Juan Miguel Mosquera, Himisha Beltran, Mark A. Rubin. Development of 2D - cell strain and 3D - tumor spheroid models for Precision Medicine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4672. doi:10.1158/1538-7445.AM2015-4672

Abstract 326: Differences in RNA expression and chemosensitivity in 2D versus 3D non-small-cell lung cancer cultures

Abstract Introduction: Increasing efforts to integrate tumour-stroma interaction and 3-dimensional structures in innovative in-vitro models were made within the last years. We established a 3D co-culture system in non-small-cell lung cancer applicable for drug testing. In this work, we present data on the differences in RNA expression and drug sensitivity in the 2D and 3D culture system. Methods: A549 and Colo699 NSCLC cell lines were both cultivated in a 2D standard cell culture (flasks, PAA, Germany) and a 3D 96 well plate hanging drop system (GravityPlusTM, Insphero, Switzerland) for 5 and 10 days, respectively. 3D microtissues were cultured either in mono- or in co-cultures with the human lung fibroblast cell line SV80. RNA expression profiling via Affymetrix chip analyses was performed with 3 biological replicates per cell line (A549, Colo699 and SV80) in mono- and co-cultures. For this purpose, RNA was isolated by a TriReagent standard protocol. Cytotoxicity was determined for cisplatin and vinorelbine after 24 hours of incubation by ATP measurements of lysed microtissues/cells and modified AnnexinV/Propidium iodide staining for flow cytometry on Colo699 cells. Furthermore, standard flow cytometry cell cycle analyses and microscopic volume measurements were performed to analyse drug induced inhibition of proliferation. Results: RNA expression profiles revealed within the A549 monocultures 892 and the Colo699 monocultures 1,042 regulated genes (≥ two-fold change in expression) for 2D versus 3D cultures, respectively. Between the cell lines A549 and Colo699 only a limited overlap (131 genes) in differentially expressed genes was detected. However, significant differences in gene expression were experienced in co-cultures with SV80. The most frequently altered genes are involved in the regulation of the cell cycle, organelle fission, RNA polymerases, histone methylation, DNA repair, oxidative stress and WNT signalling pathways. To prove the potential in-vivo relevance of these findings, we assessed the chemosensitivity in the Colo699 for 2D and 3D cell cultures. A significant difference in LD50 could be observed for cisplatin between 2D (100μM) and 3D (250μM) cultures in both ATP release measurements and AnnexinV/PI flow cytometry analyses. In cell cycle analyses the maximum M arrest for vinorelbine was achieved with 25μM in 2D, whereas in 3D 100μM of vinorelbine were needed. Cytotoxicity assays did not reveal a significant difference for vinorelbine in 2D versus 3D models. Conclusion: Herein, we demonstrate that cultivation of cell lines either in a 2D or 3D environment lead not only to a difference in RNA expression but also in chemosensitivity. Thereby, these data support the urgent need of more in vivo like cell culture models for analysing drug related modes of action. Citation Format: Gabriele Gamerith, Johannes Rainer, Arno Amann, Stefan Koeck, Edith Lorenz, Heinz Zwierzina, Julia M. Huber. Differences in RNA expression and chemosensitivity in 2D versus 3D non-small-cell lung cancer cultures. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 326. doi:10.1158/1538-7445.AM2015-326

Culturing muscle fibres in hanging drop: A novel approach to solve an old problem

The satellite cells (SCs) associated with muscle fibres play a key role in postnatal growth and regeneration of skeletal muscle. Commonly used methods of isolation and in vitro culture of SCs lead to the mixture of their subpopulations that exist within muscle. To solve this problem, we used the well established technique, the hanging drop system, to culture SCs in a three-dimensional environment and thus, to monitor them in their original niche. Using hanging drop technique, we were able to culture SCs associated with the fibre at least for 9 days with one transfer of fibres to the fresh drops. In comparison, in the classical method of myofibres culture, that is, on the dishes coated with Matrigel, SCs leave the fibres within 3 days after the isolation. Cells cultured in both systems differed in expression of Pax7 and MyoD. While almost all cells cultured in adhesion system expressed MyoD before the fifth day of the culture, the majority of SCs cultured in hanging drop still maintained expression of Pax7 and were not characterised by the presence of MyoD. Among the cells cultured with single myofibre for up to 9 days, we identified two different subclones of SCs: low proliferative clone and high proliferative clone, which differed in proliferation rate and membrane potential. The hanging drop enables the myofibres to be kept in suspension for at least 9 days, and thus, allows SCs and their niche to interact each other for prolonged time. In a consequence, SCs cultured in hanging drop maintain expression of Pax7 while those cultured in a traditional adhesion culture, that is, devoid of signals from the original niche, activate and preferentially undergo differentiation as manifested by expression of MyoD. Thus, the innovative method of SCs culturing in the hanging drop system may serve as a useful tool to study the fate of different subpopulations of these cells in their anatomical location and to determine reciprocal interactions between them and their niche.

Abstract 3836: A 3D high throughput RNA1 screen identifies the TLR4 kinase which alters spheroid architecture: a promising therapeutic strategy for pancreatic cancer.

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDA) ranks fourth among cancer-related deaths in the United States. The poor prognosis of PDA is due to resistance to current therapies. Contributing to this resistance is the tumor architecture comprising dense desmoplastic stroma, extracellular matrix (ECM), tumor cells and tumor hypoxia. We hypothesized that destabilizing the compact tumor architecture by weakening the interactions between tumor cells alone or tumor cells along with the ECM may result in more effective therapies for PDA. Methods and Results: Using the pancreatic cell line Panc1, we developed a three dimensional (3D) spheroid model system, that exhibits oxygen gradients and hypoxia that mimics the tumor microenvironment. We used the pancreatic cancer cell line PANC1-HRE that stably expresses HRE-luciferase and detects HIF-1α activation, and a platform comprising a non-matrix nano-structured scaffold, to generate compact spheroids with hypoxic inner cores and HIF-1α activation. A high throughput siRNA screen using the kinome siRNA library and our 3D spheroid modal system identified kinases whose silencing reduced both the level of hypoxia and activity of HIF-1α. The positive control, siRNA against HIF-1α, reduced activity of HIF-1α with no effect on the levels of hypoxia within the spheroids. We identified the kinases Toll-like receptor 4 (TLR4) and its downstream target spleen tyrosine kinase (SYK) as our lead candidates following target validation. Image analysis of spheroids transfected with siRNA against both TLR4 and SYK resulted in the loss of compactness of the spheroids. In addition, these spheroids also demonstrated a significant reduction in the levels of hypoxia determined by the hypoxia probe Lox-1. These findings suggest that the alteration of the spheroid architecture resulted in the reduction in hypoxia. Previous studies have shown that SYK is a direct target of TLR4. Therefore to determine the mechanism through which TLR4 dependent signaling regulates the integrity of the spheroidal architecture we are generating stable cell lines in Panc1 of shRNA targeting TLR4 under an inducible system. We will use our inducible shRNA targeting TLR4 and the two 3D model systems, the hanging drop system (analogous to a primary tumor) and the nanoculture plate system (analogous to the progression of a metastatic loci) to further confirm our findings. Conclusion: TLR4 mediated signaling regulates the integrity of spheroid architecture. Inhibiting this pathway destabilized the tumor architecture altering the hypoxic state of the spheroid. Our long-term goals are to use known pharmacological inhibitors of TLR4 to study the mechanisms by which this kinase regulates tumor architecture, determine the pre-clinical significance of targeting TLR4 utilizing autochtonous mouse models for PDA and develop TLR4-targeted therapy for PDA. Citation Format: Geoffrey A. Bartholomeusz, Alex Campos, Geoffrey Grandjean, Garth Powis. A 3D high throughput RNA1 screen identifies the TLR4 kinase which alters spheroid architecture: a promising therapeutic strategy for pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3836. doi:10.1158/1538-7445.AM2013-3836

Hanging drop: An in vitro air toxic exposure model using human lung cells in 2D and 3D structures

Using benzene as a candidate air toxicant and A549 cells as an in vitro cell model, we have developed and validated a hanging drop (HD) air exposure system that mimics an air liquid interface exposure to the lung for periods of 1h to over 20 days. Dose response curves were highly reproducible for 2D cultures but more variable for 3D cultures. By comparing the HD exposure method with other classically used air exposure systems, we found that the HD exposure method is more sensitive, more reliable and cheaper to run than medium diffusion methods and the CULTEX® system. The concentration causing 50% of reduction of cell viability (EC50) for benzene, toluene, p-xylene, m-xylene and o-xylene to A549 cells for 1h exposure in the HD system were similar to previous in vitro static air exposure. Not only cell viability could be assessed but also sub lethal biological endpoints such as DNA damage and interleukin expressions. An advantage of the HD exposure system is that bioavailability and cell concentrations can be derived from published physicochemical properties using a four compartment mass balance model. The modelled cellular effect concentrations EC50cell for 1h exposure were very similar for benzene, toluene and three xylenes and ranged from 5 to 15mmol/kgdry weight, which corresponds to the intracellular concentration of narcotic chemicals in many aquatic species, confirming the high sensitivity of this exposure method.

Failure in antigen responses by T cells from patients with common variable immunodeficiency (CVID).

Antigen-driven responses by T cells from patients with CVID and normal subjects have been assessed. Low-density cells enriched for antigen-presenting dendritic cells were cultured with T cells using a 20-microliters hanging drop system. T cells from all subgroups of CVID patients showed markedly reduced responses to the recall antigens purified protein derivative (PPD) or tetanus toxoid, whereas responses by cells from patients with X-linked agammaglobulinaemia, used as a disease control, were in the normal range. However, primary allo-stimulation of CVID T cells was normal. CVID cells from two patients failed to respond to stimulation with a neoantigen, an HIV env peptide, under conditions where normal T cells did respond. These data illustrate a profound defect in antigen-stimulated T cell proliferation in vitro in all groups of CVID patients, but do not distinguish whether the defect is in the presenting cell or in the T lymphocyte. In vivo, germinal centre B cells are thought to present antigen to primed T cells to obtain essential signals (e.g. CD40 ligand and IL-2) for B cell survival and progression to immunoglobulin secretion. A failure of antigen-specific T cell function in vivo in CVID would thus not provide the primed T cells needed for B cell rescue, and could be the primary defect leading to the low immunoglobulin production in this condition.

Initiation of placental lactogen‐I production by blastocysts growing on a two‐dimensional surface and in hanging drops

The mouse blastocyst undergoes a program of protein secretion during the perimplantation period including the initial production of placental lactogen-I (mPL-I) on day 6 of pregnancy. Although blastocysts collected on day 5 also produce mPL-I when cultured to form outgrowths on plastic dishes, it was not known if embryos have an intrinsic ability to produce mPL-I during culture in vitro, or if a specific uterine influence is necessary. Earlier experiments also suggested that attachment of the trophoblast to a stable surface may be a prerequisite for synthesis of mPL-I. Both questions were addressed by examining mPL-I production by day 3 and day 4 embryos cultured either on a two-dimensional tissue culture dish surface or in hanging drops. The presence of intracellular mPL-I was assayed by immunohistochemistry, while the secreted hormone was detected by its known position in two-dimensional electrophoresis gels. These experiments demonstrated that these earlier stage embryos do have an intrinsic program for mPL-I production which proceeds in vitro under various culture conditions. Synthesis of mPL-I occurred in embryos suspended in hanging drops as well as in those spreading on a solid two-dimensional surface, thus showing that adhesion to a surface is not required for production of this hormone. Although some mPL-I synthesis was seen in embryos cultured in medium containing BSA as the sole macromolecule, the inclusion of fibronectin either in dishes, where it supports attachment, or in the hanging drop system stimulated mPL-I secretion. Serum supplementation in both culture systems further increased growth and differentiation of the embryo, as well as mPL-I secretion, compared to fibronectin supplementation.

The uptake and localization of radioactive DOPA by amphibian melanoblasts in vitro

Neural crest explants from different anteroposterior levels of axolotl ( Ambystoma mexicanum) embryos were grown in a modified hanging drop system. The cultures were treated with 3,4-dihydroxyphenyl-alanine-α-β- 3H (DOPA- 3H) at various ages, and tritium incorporation was determined by radioautography. By day 7 after explantation, all the potential melanophores in both trunk and cranial cultures have synthesized sufficient melanin to make their positive identification possible. When treated with DOPA- 3H on day 7, 8, or 9, only cells that contain melanin incorporate labeled DOPA, and all cells that contain melanin are also radioactive. This demonstrates that definitive melanoblasts selectively incorporate DOPA, and that DOPA serves as a specific melanin precursor in these cells. Since DOPA can arise only from tyrosine, it is concluded that a tyrosine-DOPA-melanin pathway is operative during melanogenesis in amphibian pigment cells. When treated with DOPA- 3H on day 3 after explantation, one-fourth of the anterior trunk outgrowths are composed almost entirely of radioactive cells. In the remaining 3-day anterior trunk cultures, and in all the 3-day posterior trunk, anterior cranial, and posterior cranial cultures, none of the cells of any of the outgrowths are labeled. Prior to the third day, there is no incorporation of DOPA. When treated on the fourth day, or thereafter, trunk outgrowths are composed almost exclusively of radioactive cells. In the cranial outgrowths, the labeled cell population does not exceed one-third of the total. The trunk and cranial regions of neural crest are also significantly different from each other with respect to pigment cell potential in vitro. The trunk segments give rise to outgrowths which consist almost exclusively of melanophores, and the cranial segments give rise to outgrowths in which melanophores comprise less than one-third of the total cell population. The close correspondence between the frequencies of radioactive cells and the frequencies of pigment cells indicates that melanoblasts, prior to visible differentiation, selectively incorporate DOPA. The time at which presumptive pigment cells are first able to utilize exogenously administered DOPA coincides with the time at which migration ceases, indicating that the capacity to synthesize melanin is not acquired until migration has ended. The incorporation of label by the presumptive pigment cells contained in cultures 3, 4, 5, and 6 days old permits the identification of melanoblasts earlier than has previously been possible and demonstrates that these cells have developed a highly specialized biochemical system before any outward signs of it are manifest. It is clear, moreover, that amphibian melanoblasts can utilize exogenous substrate in spite of their ample internal reserves.