**6. Materials and methods**

#### **6.1 Donors and biological samples procedures**

The applied procurement protocol to vascular tissues was made on cadaver multi organ donors through informed consent and in accordance with standard operating manuals in the National Institute for Donation and Transplantation (INDT) of Uruguay. They were likewise applied legal and ethics regulations (Law 14005/1971 - 17668/2003) valid in our country. The exclusion criteria and biological safety were applied, in accordance with the Standards for Tissue Banking: International Atomic EnergyAgency (IAEA - 2005) and the

The x-rays diffractive analysis of the collagen structure was earlier studied (Pauling & Corey, 1951). Defined diffraction peak was described, in the range d = 2.86 Å (2 θ = 31.3 º using CuK α radiation λ = 1.5418 Å value), based on the criteria of the Bragg Law. This phenomenon was interpreted as the expression of cis configurations for the amide groups of

Using this background, our group analyzed tissue banking allograft, in order to compare the diffraction profiles obtained before and after cryo-preservation method (vascular tissues and amnion tissue), and glycerolized preserved method (amniotic membrane). The working hypothesis states that the preservation methods can modify the stereochemistry molecular structures, determining changes in collagen and the consequent differentiation of diffractive

The applied procurement protocol to vascular tissues was made on cadaver multi organ donors through informed consent and in accordance with standard operating manuals in the National Institute for Donation and Transplantation (INDT) of Uruguay. They were likewise applied legal and ethics regulations (Law 14005/1971 - 17668/2003) valid in our country. The exclusion criteria and biological safety were applied, in accordance with the Standards for Tissue Banking: International Atomic EnergyAgency (IAEA - 2005) and the

Fig. 2. Raman Spectum oleic acid

the polypeptide chain.

I N T E N S I T Y A.U

or dispersive profile.

**6. Materials and methods** 

**6.1 Donors and biological samples procedures** 

**5.4 Interaction x-rays - Collagen: A model analysis** 

Spanish Association of Tissue Banks (AEBT - 2005). The same selection criteria, exclusion, tissue procurement and processing were applied to living donor placenta with clinical controlled normal pregnancy and delivery, by the parameters set by the Ministry of Public Health of Uruguay.

Donors were selected according to the protocol in a range between 18 and 60 years (35.5 ± 11.8 years mean age, 47% M 53% F) obtained by aseptic dissection, 10 aortic arterial segments, and 8 carotid. It proceeded under a laminar flow cabinet to cleaning, package, and storage in physiological saline at 4°C. Fresh Vascular Samples (FVS) were shipped to DETEMA within 24 hs, for XRD. The same process aseptic protocol was applied to 6 amnion obtained by manual dissection from donor placenta. Fresh Amnion Samples, (FAS) were stored in saline solution at 4ºC and shipping to DETEMA within 24 hs for XRD analysis.

The Cryopreserved Vascular Samples (CVS), segments of each contra lateral carotid donor, and hemi ring segments of thoracic descendent aorta, were processed for cryopreservation in a Controlled Rate Freezing System (Model 9000, Gordinier Electronics, Inc. Michigan). Stored CVS were maintained up to 30 days at -142ºC. The cryopreservation media was: RPMI 1640, 85 cc; Human Albumin (20%), 5 cc; DMSO 10%. Cryopreservation was made into termal sealed double cryo resistant bag (Joisten and Kettenbaum D51429, Bereisch Gladbach, Mod.011342). The mean cooling rate applied was -1ºC/min from 4ºC to -90ºC and then quickly stored at -142ºC during 30 days in steam liquid nitrogen.

Same procedures were applied to obtain 2 Cryopreserved Amnion Samples (CAS), stored up to 30 days at -142ºC.

Defrost protocol applied for vascular and amniotic tissues, was according with Pegg et al. (1997), and defrosted samples were shipped to DETEMA for XRD.

6 Glycerolized Amnion Samples (GAS) were obtained by soaked in screw cap flask in Glycerol (95%) and stored at 4º C for 30 days. Glycerol from amnion was removed by three sequential shaking washing for 15 min. each, in saline solution and then shipped to DETEMA for x-rays diffraction, and Raman Scattering analysis. The comparative assays were done with FVS vs CVS; FAS vs CAS; and FAS vs GAS.
