**1. Introduction**

68 Current Frontiers in Cryopreservation

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Bones and tendons are obtained from donors who have been pronounced brain dead after a rigid and extensive screening process. The tissues obtained are sent to a Tissue Bank and submitted to processing steps, packaging and cryopreservation at -80◦C. It is vital to maintain sterility and integrity, so that no tissue is discarded.These precautions also extend to the packaging, which should promote containment and protection. Although minimum technical criteria have already been defined for the food industry, this has still not been regularized in Brazil. We emphasize, therefore, the need to study this subject, focusing on maintaining the quality of the musculoskeletal tissues produced by tissue banks. All procedures developed byTissue Banks currently present in the country have rigid control over the quality and the traceability of tissues made available, based on international standards (EATB, 2004; AATB, 2007) on the legislation (BRASIL, 1997-2006) and in conformity with Good Manufacturing Practices - GMP.

### **2. Cryopreservation of musculoskeletal tissues**

In the cryopreservation room, the tissues are stored according to their status in the process. Thus, there are designated areas for tissues under analysis or in quarantine (where they remain for around 60 days before the result of all the exams) and for those that have been liberated for use. both areas are equipped with ultra-low temperature freezers, with temperatures ranging from minus 80 to minus 100 degrees Celsius.

*<sup>1</sup>Institute of Orthopedics and Traumatology, Hospital das Clínicas of the University of São Paulo School of Medicine, Sao Paulo, SP, Brazil* 

*<sup>2</sup>Packaging Technology Center – Institute of Food Technology, Campinas, SP, Brazil 3Nuclear and Energy Research Institute - IPEN/CNEN-SP – São Paulo, Brazil 4Biomechanics Laboratory - Institute of Orthopedics and Traumatology, Hospital das Clínicas of the University of São Paulo school of Medicine, Brazil* 

*<sup>5</sup>Dental Student Institute of Orthopedics and Traumatology, Hospital das Clínicas of the University of São Paulo school of Medicine, São Paulo, SP, Brazil* 

*<sup>6</sup>National Health Surveillance Agency- ANVISA*\*

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues 71

Layer 02: LLDPE – 1-octene comonomer Linear Low-Density Polyethylene and 1-octene

Layer 04: LLDPE – 1-octene comonomer Linear Low-Density Polyethylene and 1-octene

Layer 06: LLDPE – 1-octene comonomer Linear Low-Density Polyethylene and 1-octene

In relation to the physicomechanical and barrier properties characteristics, we investigated the alterations that occurred in the packages after 30, 60, 90, 120, and 150 days of cryopreservation at -80◦C. For the cytotoxicity and sterility analysis, two groups (before and

**A) Characterization of coex film in relation to its thickness and water vapor transmission** 

The thickness of each layer of plastic material in the film sample was determined through images captured by a Metaval inverted microscope operating at a magnification of 200x, using the image analysis system Axio Vision (Zeiss®). The cross-section of the sample was obtained using a Leica microtome, model RM2245, with section thickness set to 40 μm. To facilitate visualization of the barrier layer, 2% iodine solution was used as a contrasting agent. Five cross-section samples of the material were obtained; five measurements were performed for each sample, totalling 25 thickness measurements. The test was carried out at a temperature of around 23°C, after packaging of the sample in a controlled environment at 23°C ± 2°C and (50 ± 3)% relative humidity for a minimum period of 48

The water vapor transmission rate was determined using a MOCON PERMATRAN-W 3/31 device, following the procedure described in regulation ASTM F1249-06 - Standard test methods for water vapor transmission rate through plastic film and sheeting using a modulated infrared sensor. In this test, the water vapor that passes through the film is carried to the infrared sensor by ultra-dry nitrogen flow. The sensor measures the fraction of energy absorbed by the water vapor, and emits an electrical signal with amplitude proportional to the concentration of water vapor. The range of this signal is compared with that of the signal produced by the water vapor that passes through a calibration film with a known water vapour transmission rate. The effective permeation area of each sample was 50cm2. The assay was performed at 38oC/100%RH and in this condition, the calibration standard showed water vapour transmission rate of 4.54g water.m-2.day-1. The water vapor transmission rate of the sample was corrected for the condition 38oC/90%RH, multiplying

The total thickness of each layer of coextruded film is shown in Table 1. Figure 10 shows an

example of a cross-section image of the sample obtained for the assay.

Layer 01: LLDPE – 1-octene comonomer Linear Low-Density Polyethylene

comonomer Linear Low-Density Polyethylene modified with anhydride maleic Layer 03: PA – hexamethylenediamine, adipic acid e caprolactam copolyamide

comonomer Linear Low-Density Polyethylene modified with anhydride maleic Layer 05: PA – hexamethylenediamine, adipic acid e caprolactamcopolyamide

comonomer Linear Low-Density Polyethylene modified with anhydride maleic Layer 07: LLDPE – 1-octene comonomer Linear Low-Density Polyethylene

after sterilization by ethylene oxide) were analysed.

**rate (WVTR)** 

hours.

the results by a factor of 0.9.

**3.2 Physicomechanical and barrier properties tests** 

The room is also equipped with an air conditioning system, its own energy generator, and carbon gas backup as protection against defrosting, as well as a rigorous temperature monitoring system that generates 24-hour printed temperature recordings, and a satellite alarm system, which ensures adequate maintenance of the temperature and early detection of any flaws.

Depending on the results of the analyses, the tissues are transferred to the room designated for materials liberated for use. The maximum cryopreservation period is 5 years for bone tissue and 2 years for soft or tendinous tissues.

It is vital to maintain sterility and integrity, in order to avoid disposal of any material. This is the purpose of **packaging**, which is aimed at containment and protection. In Brazil, there are no specific regulations for packaging of sterile, cryopreserved tissues for transplants, and in this chapter, we present our experience in the definition and validation of a type of packaging used for this purpose. Our proposal is to characterize a coextruded plastic film structure used for packaging of musculoskeletal tissues at low temperatures, in regard to the aspects sterility, cytotoxicity, migration, mechanical resistance and oxygen permeability.

Fig. 1. Cryopreservation of tissues at -80 ◦C in drawers, separated by batches.
