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Isolation, purification and in vitro differentiation of cytotrophoblast cells from human term placenta.

Li L, Schust DJ - Reprod. Biol. Endocrinol. (2015)

Bottom Line: Isolated cytotrophoblast cells began to aggregate into monolayers of mononucleated cells within about 12 h of plating.By 72 h in culture, most cytotrophoblast cells have differentiated into syncytiotrophoblast as demonstrated by a loss of intercellular E-cadherin expression upon fusion into multinucleated syncytia.We present an efficient and reliable method for isolating of cytotrophoblast cells with high purity and complete differentiation into syncytiotrophoblast in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics, Gynecology and Women's Health, University of Missouri School of Medicine, 500 N. Keene Street, Columbia, MO, USA. doctorlipingli@foxmail.com.

ABSTRACT

Background: The syncytialization of cytotrophoblast cells to syncytiotrophoblast is central to human placental transport and hormone production. Many techniques for in vitro study of this process have been proposed and new investigators to the field may find the literature in the field daunting. Here, we present a straightforward and reliable method to establish this important model using modern but readily available tools and reagents.

Methods: Villous cytotrophoblast cells are obtained from term placenta using mild enzymatic degradation, Percoll gradient centrifugation, negative magnetic cell sorting using an antibody against classical major histocompatibility complex molecules and in vitro culture on a matrix-coated growth surface.

Results: The purity of isolated cytotrophoblast cells exceeds 98 % as assessed by cytokeratin-7 expression using flow cytometry. Contamination by mesenchymal cells, extravillous trophoblast cells, leukocytes, Hofbauer and endothelial cells is minimized (less than 2 % when analyzed for vimentin, HLA-G, CD45, CD163 and CD31 using flow cytometry). Isolated cytotrophoblast cells began to aggregate into monolayers of mononucleated cells within about 12 h of plating. By 72 h in culture, most cytotrophoblast cells have differentiated into syncytiotrophoblast as demonstrated by a loss of intercellular E-cadherin expression upon fusion into multinucleated syncytia. After 72 h in culture, nearly every cultured cell expresses syncytiotrophoblast markers, including cytokeratin-7, human chorionic gonadotropin-β (β-hCG) and the fusion-related proteins glial cell missing-1 (GCM-1) and syncytin.

Conclusions: We present an efficient and reliable method for isolating of cytotrophoblast cells with high purity and complete differentiation into syncytiotrophoblast in vitro.

No MeSH data available.


Related in: MedlinePlus

Comparison of yield and viability of purified cytotrophoblast cells among a variety of enzymatic degradation protocols. Yield (a) and viability (b) of purified cytotrophoblast cells using different enzymatic digestion protocols were assessed. Protocol 1: digestion three times in 0.25 % trypsin for 30 min each [10]; Protocol 2: digestion two times in 0.25 % trypsin for 10 min each [11]; Protocol 3: digestion in 0.125 % trypsin and 0.2 mg/ml DNase I for 45 min [14]; Protocol 4: digestion three times in 0.125 % trypsin and 0.2 mg/ml DNase I for 30 min each [15]; Protocol 5: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 15 min each; Protocol 6: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 20 min each; Protocol 7: digestion two times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 30 min each. Data are presented as mean ± SD of six independent experiments
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Fig1: Comparison of yield and viability of purified cytotrophoblast cells among a variety of enzymatic degradation protocols. Yield (a) and viability (b) of purified cytotrophoblast cells using different enzymatic digestion protocols were assessed. Protocol 1: digestion three times in 0.25 % trypsin for 30 min each [10]; Protocol 2: digestion two times in 0.25 % trypsin for 10 min each [11]; Protocol 3: digestion in 0.125 % trypsin and 0.2 mg/ml DNase I for 45 min [14]; Protocol 4: digestion three times in 0.125 % trypsin and 0.2 mg/ml DNase I for 30 min each [15]; Protocol 5: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 15 min each; Protocol 6: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 20 min each; Protocol 7: digestion two times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 30 min each. Data are presented as mean ± SD of six independent experiments

Mentions: To determine which digestion protocol simultaneously optimized yield and viability in the isolated cytotrophoblast cells, we exposed placental tissue to trypsin alone [10, 11] or in combination with other enzymes [14, 15], as well as proteolytic enzymes other than trypsin for various incubation times. As shown in Fig. 1, three digestions for 20 min each using an enzymatic cocktail composed of dispase II, collagenase I and DNase I (Protocol 6) resulted in the best combination of yield and cell viability. Using this protocol, the average yield of purified cytotrophoblast cells was (1.11 ± 0.07) × 106 cells/gram tissue and average cell viability was 94.4 % ± 3.2 % as judged by Trypan blue exclusion (n = 6).Fig. 1


Isolation, purification and in vitro differentiation of cytotrophoblast cells from human term placenta.

Li L, Schust DJ - Reprod. Biol. Endocrinol. (2015)

Comparison of yield and viability of purified cytotrophoblast cells among a variety of enzymatic degradation protocols. Yield (a) and viability (b) of purified cytotrophoblast cells using different enzymatic digestion protocols were assessed. Protocol 1: digestion three times in 0.25 % trypsin for 30 min each [10]; Protocol 2: digestion two times in 0.25 % trypsin for 10 min each [11]; Protocol 3: digestion in 0.125 % trypsin and 0.2 mg/ml DNase I for 45 min [14]; Protocol 4: digestion three times in 0.125 % trypsin and 0.2 mg/ml DNase I for 30 min each [15]; Protocol 5: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 15 min each; Protocol 6: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 20 min each; Protocol 7: digestion two times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 30 min each. Data are presented as mean ± SD of six independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4497497&req=5

Fig1: Comparison of yield and viability of purified cytotrophoblast cells among a variety of enzymatic degradation protocols. Yield (a) and viability (b) of purified cytotrophoblast cells using different enzymatic digestion protocols were assessed. Protocol 1: digestion three times in 0.25 % trypsin for 30 min each [10]; Protocol 2: digestion two times in 0.25 % trypsin for 10 min each [11]; Protocol 3: digestion in 0.125 % trypsin and 0.2 mg/ml DNase I for 45 min [14]; Protocol 4: digestion three times in 0.125 % trypsin and 0.2 mg/ml DNase I for 30 min each [15]; Protocol 5: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 15 min each; Protocol 6: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 20 min each; Protocol 7: digestion two times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 30 min each. Data are presented as mean ± SD of six independent experiments
Mentions: To determine which digestion protocol simultaneously optimized yield and viability in the isolated cytotrophoblast cells, we exposed placental tissue to trypsin alone [10, 11] or in combination with other enzymes [14, 15], as well as proteolytic enzymes other than trypsin for various incubation times. As shown in Fig. 1, three digestions for 20 min each using an enzymatic cocktail composed of dispase II, collagenase I and DNase I (Protocol 6) resulted in the best combination of yield and cell viability. Using this protocol, the average yield of purified cytotrophoblast cells was (1.11 ± 0.07) × 106 cells/gram tissue and average cell viability was 94.4 % ± 3.2 % as judged by Trypan blue exclusion (n = 6).Fig. 1

Bottom Line: Isolated cytotrophoblast cells began to aggregate into monolayers of mononucleated cells within about 12 h of plating.By 72 h in culture, most cytotrophoblast cells have differentiated into syncytiotrophoblast as demonstrated by a loss of intercellular E-cadherin expression upon fusion into multinucleated syncytia.We present an efficient and reliable method for isolating of cytotrophoblast cells with high purity and complete differentiation into syncytiotrophoblast in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics, Gynecology and Women's Health, University of Missouri School of Medicine, 500 N. Keene Street, Columbia, MO, USA. doctorlipingli@foxmail.com.

ABSTRACT

Background: The syncytialization of cytotrophoblast cells to syncytiotrophoblast is central to human placental transport and hormone production. Many techniques for in vitro study of this process have been proposed and new investigators to the field may find the literature in the field daunting. Here, we present a straightforward and reliable method to establish this important model using modern but readily available tools and reagents.

Methods: Villous cytotrophoblast cells are obtained from term placenta using mild enzymatic degradation, Percoll gradient centrifugation, negative magnetic cell sorting using an antibody against classical major histocompatibility complex molecules and in vitro culture on a matrix-coated growth surface.

Results: The purity of isolated cytotrophoblast cells exceeds 98 % as assessed by cytokeratin-7 expression using flow cytometry. Contamination by mesenchymal cells, extravillous trophoblast cells, leukocytes, Hofbauer and endothelial cells is minimized (less than 2 % when analyzed for vimentin, HLA-G, CD45, CD163 and CD31 using flow cytometry). Isolated cytotrophoblast cells began to aggregate into monolayers of mononucleated cells within about 12 h of plating. By 72 h in culture, most cytotrophoblast cells have differentiated into syncytiotrophoblast as demonstrated by a loss of intercellular E-cadherin expression upon fusion into multinucleated syncytia. After 72 h in culture, nearly every cultured cell expresses syncytiotrophoblast markers, including cytokeratin-7, human chorionic gonadotropin-β (β-hCG) and the fusion-related proteins glial cell missing-1 (GCM-1) and syncytin.

Conclusions: We present an efficient and reliable method for isolating of cytotrophoblast cells with high purity and complete differentiation into syncytiotrophoblast in vitro.

No MeSH data available.


Related in: MedlinePlus