Vascular Regulation by endothelial Cells: Comparison
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Please note this is a comparison between Version 2 by Dean Liu and Version 1 by Elisa Cairrao.

Human umbilical cord (HUC) is a suitable source for isolation of endothelial cells (ECs) since it has no particular ethical impediments and is considered a non-tumorigenic and less immunogenic model. for this reason, HUC represents an advantageous experimental source for the isolation of endothelial cells. The ECs can produce/release molecules that modulate vasoconstriction and vasorelaxation by smooth muscle cells (SMC).

  • endothelial cells
  • cardiovascular system
  • umbilical vein
  • umbilical artery
  • smooth muscle cells

1. Introduction

Cardiovascular diseases (CVD) constitute the major cause of death worldwide, with a higher prevalence in the adult population [1,2][1][2]. Examples of CVD are coronary artery disease, peripheral arterial disease, or aortic disease.

Currently, the human umbilical cord (HUC) is a suitable source for isolation of endothelial cells (ECs) since it has no particular ethical impediments and is considered a non-tumorigenic and less immunogenic model. for this reason, HUC represents an advantageous experimental source for the isolation of endothelial cells [6][3]. The ECs can produce/release molecules that modulate vasoconstriction and vasorelaxation by smooth muscle cells (SMC) [7,8][4][5]. When releasing nitric oxide (NO), prostacyclin, bradykinin, and endothelium-derived hyperpolarizing factor (EDHF), the response given will be vasodilation by the SMC. If the substances released are endothelin-1 (ET-1), angiotensin II (Ang II), thromboxane A2 (TXA2), the final effect is vasoconstriction by the SMC [9][6].

The effect of endothelium in vivo is very difficult to study. To counteract this, several in vitro techniques were developed. Among these techniques, the use of human umbilical vein endothelial cells (HUVECs) was widely used as a source of human endothelial cells because they have some advantages. The main advantage is that it is obtained from non-pathogenic human tissues, which is not the case with other human sources, and the physiological relevance in the control of vascular pathways. Moreover, primary cultures with these cells can maintain native characteristics of endothelial cells and their intracellular signaling pathways [10][7].

2. Morphological Characteristics of the Human Umbilical Cord (HUC)

From a physiological point of view, the human umbilical cord (HUC) is a channel developed from the amniotic sac (which forms the epithelium), the allantois (which forms the umbilical veins and arteries), and the yolk sac [12,13][8][9].

Thus, not only the number of blood vessels but also morphological changes can contribute to the development of pathologies. We can conclude that the presence or absence of blood vessels is of extreme importance for the development of pathology.

In summary, not only the number of blood vessels but also morphological changes can contribute to the development of pathologies.

Finally, HUA does not have vasa vasorum or a nervi vasorum, nor a typical external tunica (adventitia). The function of this tunica is performed by Wharton’s jelly (mucous connective tissue and fibroblasts) which is surrounded by nutritional vessels and rich in glycosaminoglycans [12,16,20][8][10][11].

3. Isolation of Endothelial Cells of HUC

During the last 3 decades, advances in vascular endothelial cell biology have had a great impact on the understanding of some pathophysiological conditions such as cardiovascular diseases [35,36][12][13]. Between 1922 and 1973, there was a growing interest among the scientific community in human umbilical cord vein-derived endothelial cells (HUVEC) for these investigations [1]. In 1922, studies were conducted and described the development of endothelial cells from chicken liver embryonic tissue explants [37][14].

In 1973, Eric A. Jaffe successfully isolated HUVEC, which grew as a homogeneous monolayer of polygonal cells with well-defined borders. Furthermore, these authors also compared HUVECs with SMC and fibroblasts, which allowed them to conclude that HUVECs contained rod-shaped cytoplasmic organelles [39][15]. In this procedure, veins were perfused with collagenase and incubated for 15 min at 37 °C, and endothelial cells were collected at the end [39][15].

Since 1974, some studies of isolation of EC have appeared intending to study the physiology of EC, most of them based on the studies and descriptions made by the following authors [11,41,42,43][16][17][18][19]. Over time, these studies have undergone some adaptations, such as the constitution of the culture medium for endothelial cells. In this sense, the constitution of the culture medium used may contain endothelial cell growth factor (ECGF), which allows long-term growth of the ECs, or an anti-PPLO agent that prevents contamination by gram-positive bacteria or mycoplasmas [41,44,45,46,47][17][20][21][22][23]. Culture media containing 5 ng/mL epidermal growth factor (EGF) was shown to have a higher proliferative potential compared to the anti-pleuropneumonia-like organisms’ agent (anti-PPLO agent).

Meanwhile, some authors performed with the same umbilical cord the culture of HUVECs and the culture of HUAECs, and observed that cell isolation by enzymatic digestion is easy and fast when the artery and vein are used from a single umbilical cord [42][18]. Furthermore, other authors performed the isolation of the HUVECs and the HUCMSC (umbilical cord mesenchymal stem cells) from the same umbilical cord [57][24], as shown in Table 1 . The culture of HUCMSC may also allow us to direct the fate towards an endothelial cell lineage, which can represent economical and commercially viable option manly for cell replacement therapy due to its noninvasive collection procedure [57][24].

Table 1.
 Conditions used for isolation of endothelial cells.
Studies

Performed		 Digestive Enzyme Medium Used Centrifugation Conditions Coating Cells Protocol Based on Observations
Maruyama et al. (1963) [38][25] 0.2% Trypsin YLH - Rat-tail Collagen (coverslip) HUVECs - The cells were cultured in sheets
Jaffe et al. (1973) [39][15] 0.2% Collagenase TC 199 250 g

10 min		 - HUVECs Based on

Maruyama et al.		 ECs differentiation based on morphologic and immunologic criteria
Mann et al. (1989) [65][26] Collagenase type II M199 - 1% Gelatin HUVECs Based on

Jaffe et al.		 ECs were cultured on microcarrier beads
Sobrevia et al. (1995) [66][27] Collagenase M199 - - HUVECs - HUC from gestational diabetic pregnancies
Marin et al. (2001) [41][17] Collagenase A M199 1500 r/min

5 min		 1% Gelatin HUVECs - Three different cords were used to limit the variability of ECs
Ulrich-mersenich et al. (2002) [42][18] Dispase II and Collagenase tipo IV M199 1500 rpm

15 min		 Fibronectin HUVECS and HUAECS Based on

Ko et al. (1995)		 Isolation of ECs and SMC from the same vessel of HUC obtain the best results
Larrivee et al. (2005) [48][28] Collagenase A MCDB 131 300 g

5 min		 0.2% Gelatin HUVECs - ECs successful cryopreservation
Mahabeleshwar et al. (2006) [67][29] 0.1% Collagenase Endothelial cell growth medium 320 g

10 min		 Gelatin HUVECs - Obtention pure cultures of ECs
Baudin et al. (2007) [45][21] 0.2% Collagenase M199 750 g

10 min		 Fibronectin HUVECs Based on

Jaffe et al.		 HUVECs can keep phenotypic features in free serum medium for up 12 h
Crampton et al. (2007) [68][30] Collagenase M199 1200 rpm

5 min		 Gelatin HUVECs - Used in angiogenesis assay
Cheung et al. (2007) [46][22] 0.1% Collagenase M199 250 g

10 min Room temperature		 Gelatin HUVECs - Confluent T25 flask was obtained in 4–5 days. Cells beyond the 6th passage should be discarded.
Martin de llano et al. (2007) [11][16] Collagenase type I and Dispase M200 or M231 10 g

10 min		 Fibronectin HUVECs and HUAECs - Birth weight does not have influence on the time request to obtain grown cultures
Casanello et al. (2009) [69][31] Collagenase M199 - - HUVECs Based on

Sobrevia et al.		 ECs maintain the

protein expression until 5th passage
Kadam et al. (2009) [57][24] Dispase II and Collagenase type IV M199 1500 rpm

15 min		 Fibronectin HUVECS and hUCMSCs Based on Ulrich-mersenich et al. Two-steps protocol for the simultaneous isolation of ECs and hUCMSCs
Krause et al. (2012) [70][32] Collagenase M199 - - HUVECs and HUAECs Based on

Casanello et al.		 ECs are responsive to hypoxia
Pang et al. (2012) [71][33] Collagenase M199 1000 g

5 min		 - HUVECs Based on

Jaffe et al.		 ECs are responsive to cytokine stimulation
Siow et al. (2012) [36][13] Collagenase M199 1000 rpm

5 min		 Gelatin HUVECs Based on

Jaffe et al.		 Confluent culture of ECs after 2–3 days will start to detach and decrease viability
Lattuada et al. (2013) [72][34] 0.1% Collagenase A M199 463 g

15 min		 - HUVECs Based on

Jaffe et al.		 Gravitational field-flow fractionation is the new method to easy isolate ECs from HUC
Lei et al. (2016) [73][35] 0.2% Collagenasee M199 800 g

5 min		 Gelatin HUVECs - Used a new instrument for insertion of HUC
Krause et al. (2016) [74][36] Collagenase M199 - - HUAECs Based on Krause et al. (2012) ECs pure culture can be used for protein expression
Amrithraj et al. (2017) [51][37] Collagenase EGM-2 1200 rpm

5 min		 Gelatin HUVECs Based on Crampton et al. ECs culture from gestational diabetic pregnancies maintain metabolic and molecular imprints of maternal hyperglycemia
Brodowaski et al. (2017) [50][38] 0.2% Collagenase EGM - - HUVEC Based on

Jaffe et al.		 EC culture from preeclamptic women
Di tomo et al. (2017) [55][39] Collagenase 1 M199 - 1.5% Gelatin HUVECs - EC culture was obtained with explants
Suhaila et al. (2017) [75][40] Collagenase type I M199 1500 rpm

5 min		 They tested various concentrations of gelatin HUVECs - The 0.2% gelatin obtain the best results
Thormodsson et al. (2018) [76][41] Collagenase M199 140 g

5 min		 - HUVECS and HUAECs - Using NunclonTM Δ T-25 flasks: the cells attach without any problem
Yang et al. (2018) [43][19] 0.05% Collagenase I EGM-2 250 g

5 min		 Fibronectin HUVECs Based on

Jaffe et al.		 The cells were negative for CD34, CD45, and human leukocyte antigen–DR isotype (HLA-DR)
Provitera et al. (2019) [54][42] 0.1% Collagenase A M199 463 g

15 min		 - HUAECs and

HUVECs		 Based on

Lattuada et al.		 Cells were used at P0
Pipino et al. (2020) [49][43] Collagenase type1A DMEM - 1.5% Gelatin HUVECs Based on

Di tomo et al.		 Cells between the 3rd and 7th passages were used
Psefteli et al. (2021) [52][44] 0.2% Collagenase M199 - Gelatin HUVECs Based on

Jaffe et al.		 Cells between P0 and P3 were used for protein expression

 

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